BACKGROUND
thyroid hormones  are important signaling molecules in vertebrates that regulate homeostasis, growth, and development.
one developmental process that is dependent upon the presence of th is amphibian metamorphosis.
during metamorphosis the larval, aquatic, herbivorous tadpole transforms into a terrestrial, carnivorous juvenile frog.
this event requires drastic changes in essentially every organ and tissue of the tadpole and includes: the resorption of larval organs and tissues, remodeling of larval organs into juvenile form, and de novo development of organs and tissues  <cit> .
metamorphosis is completely controlled through the control of serum th levels.
the thyroid gland mainly produces the thyroid hormone,  <dig> ,3',5'-tetraiodothyronine , which is converted into the biologically more active form,  <dig> ,3'-triiodothyronine , in the peripheral tissues  <cit> .
progression through natural metamorphosis is dependent upon a tightly-controlled rise and fall in th levels.
premetamorphic tadpoles are functionally athyroid with no measurable levels of ths  <cit> .
th levels gradually increase during prometamorphosis and reach maximal levels at metamorphic climax.
at this stage, overt remodelling of the tadpole rapidly ensues.
premetamorphic tadpoles can be induced to undergo precocious metamorphosis by exposure to th  <cit> .
the best understood mechanism of th action involves th binding to nuclear thyroid hormone receptors  which regulate gene expression  <cit> .
th binding to trs can either activate or repress transcription of responsive genes through recruitment of coactivators and corepressors, respectively  <cit> .
differential nuclear tr levels and intracellular t <dig> levels controlled by deiodinase activity and th-binding proteins, contribute to tissue-specific responses  <cit> .
however, the response to th also depends on the existing complement of other proteins that can influence cell fate .
however, the molecular mechanisms are poorly understood.
most th-responsive genes are up-regulated and these have been most commonly studied particularly in the tail  <cit> .
the genetic program required for tail regression is established between  <dig> and  <dig> h of th exposure at a "commitment point" after which removal of th or exposure to transcription or protein synthesis inhibitors cannot prevent regression  <cit> .
studies based on pcr subtractive hybridization methods and cdna gene arrays have identified a number of possible genes involved in this process  <cit> .
however, the relationship of most of these genomic findings to changes in the proteome has yet to be identified and there is growing evidence for non-classical th action through phosphorylation signaling pathways  <cit> .
proteomic scale studies on th-dependent tail regression have been scarce.
ray et al.
identified several 35s-methionine labelled proteins from rana catesbeiana tail fin that change during natural and precocious metamorphosis using two-dimensional  gel analysis  <cit> .
kobayashi et al.
used 2d gel electrophoresis to analyze changes in protein expression in the back and tail skin of xenopus laevis during metamorphosis  <cit> .
from the 2d protein spot patterns they could classify the back skin into larval or adult type and observe the transition.
attempts were made in these studies to identify the altered protein spots.
this, however, involved identifying the spots based on position, comigration or immunological detection methods.
using 2d gel separation and mass spectrometry  for protein identification, we were able to identify  <dig> proteins whose expression was altered in the x. laevis tadpole tail during th-induced metamorphosis  <cit> .
regardless of the approach, the lack of any sample fractionation led to the identification of only abundant proteins.
in this work we identified novel changes in the proteome and phosphoproteome associated with the induction of t3-dependent tail regression of rana catesbeiana tadpoles.
proteins that changed in abundance were detected using two-dimensional  gel electrophoresis and isobaric tags for relative and absolute quantitation  methods.
alterations in phosphorylation were revealed using a phosphoprotein-specific stain.
mass spectrometry  analyses were then used for protein identification.
proteome coverage was enhanced through the use of cell and protein fractionation to reveal several proteins that are altered within  <dig> h of t <dig> exposure.
RESULTS
fractionation of the tail fin proteome and 2d gel analyses
the subcellular fractionation method was optimized to provide highly enriched fractions of cytosolic, mitochondrial, nuclear and microsomal samples from the r. catesbeiana tail fin.
the goals of the method were to limit sample complexity with effective cell fractionation with minimal cross-contamination and reasonable yields.
the fractions also had to be salt and buffer-compatible with subsequent proteomic analysis.
we developed two separate procedures based on differential centrifugation to generate nuclear and cytosolic/mitochondrial/microsomal fractions .
the nuclear extraction procedure was optimized to minimize nuclear clumping, increase nuclei stability, and minimize cytoskeletal, cytoplasmic/organelle and dna contamination.
the cytosolic/mitochondrial/microsomal extraction procedure was developed to increase the disruption of tail fin cell membrane and increase mitochondrial stability and purity.
the mitochondria-enriched fraction was obtained with a  <dig>  × g centrifugation and it likely also contained lysosomes, peroxisomes, golgi and endoplasmic reticulum .
centrifugation at  <dig>  × g removed the vesicles of the plasma membrane, endosomes, golgi and er into the microsomal pellet leaving the cytosolic supernatant with its soluble molecules .
nuclear integrity was monitored by microscopy  and fractionation efficiency was determined using immunoblot analysis for subcellular markers.
an immunoblot for cytochrome c  shows substantial enrichment for that organelle in the mitochondrial fraction  while an immunoblot for the nuclear markers, lamins b <dig> and b <dig>  shows enrichment of nuclei in the nuclear fraction .
the cytosolic fraction is a complex mixture of many proteins and was therefore further fractionated using anion-exchange high performance liquid chromatography  .
a step-gradient anion-exchange hplc procedure was developed that used ammonium bicarbonate as a volatile buffer in place of commonly used salt and non-volatile buffer to provide salt-free fractions after lyophilization to render the samples compatible with the subsequent 2d gel analysis.
the cytosolic fraction was thus further fractionated into five fractions:  <dig> mm ,  <dig> mm,  <dig> mm,  <dig> mm and  <dig> m ammonium bicarbonate with each fraction  yielding roughly equal amounts of protein as shown by sds-page .
proteins within each of the resulting fractions were then separated by 2d polyacrylamide gel electrophoresis which separates proteins based on their molecular weight and isoelectric point .
therefore, the entire fractionation protocol divided the tail fin proteome over eight 2d gels: nuclear, mitochondrial, microsomal  and five cytosolic fractions .
this fractionation method increases the ability to observe expression changes in low abundance proteins and provides information on subcellular localization of proteins which cannot be achieved by examining a whole cell homogenate on a single 2d gel.
from our results, it is evident that each of the fractions shows a distinctive pattern of spots with many unique spots per fraction analyzed .
there is some overlap with the more abundant protein spots between the neighboring fractions of the hplc separation and between the microsomal and mitochondrial fractions, which probably share many cellular membrane compartments .
phosphoproteins were detected on the 2d gels with a phosphoprotein-specific fluorescent stain  which detects phosphorylation on ser, thr and tyr residues  <cit>  .
total proteins and phosphoproteins were detected in the same 2d gel allowing for easy identification of phosphoprotein location and subsequent isolation for ms analysis.
the above methods were used to analyze the proteome and phosphoproteome of the premetamorphic r. catesbeiana tail fin undergoing precocious metamorphosis at  <dig> and  <dig> h induced with  <dig> nm t <dig>  a minimum of three independent replicates allowed for the verification of changes in protein and phosphoprotein expression and ms analysis was used for protein identification.
identification of a unique r. catesbeiana keratin fragment
a prominent protein spot at ~ <dig> kda and pi ~ <dig> was increased upon t <dig> treatment on the 2d gels of several fractions .
it was observed in the  <dig> mm cytoplasmic fraction as well as in the microsomal, mitochondrial and nuclear fractions.
this protein spot was increased by 2– <dig> fold as early as  <dig> h , but was more intensely expressed at  <dig> h  .
the greatest increase was observed in the microsomal fraction.
the protein spots from each of the fractions were separately analyzed by mass spectrometry proving that each fraction represented the same protein.
a combination of electrospray-ionization quadrupole time-of-flight  and matrix-assisted laser desorption ionization tof-tof  tandem-ms  analyses allowed for peptide sequence information to be obtained for  <dig> different peptides from this protein .
protein database searches with these peptides gave the highest homology match to the x. laevis type i keratin  <dig> kda protein  also known as x. laevis keratin b <dig> [ncbi: 1304283b) from the xk <dig> gene family  <cit> .
1observed peptide masses resulting from the tryptic digestion of the protein spot, reported as singly charged.
2peptide sequence information deduced from ms/ms spectra of the corresponding peptides from esi-qqtof analysis.
the masses of isoleucine are indistinguishable from leucine in ms and therefore l can be i and vice versa.
3indicates which peptides were additionally observed with maldi-tof-tof analysis.
4percent confidence for the peptide sequences as reported by peaks software for the esi-qqtof spectra and by mascot for maldi-tof-tof data.
5highest homology match from protein database searching with the observed peptide sequences to x. laevis type i keratin  <dig> kd using spider software.
bold lettering indicates differences between the observed and database sequences.
parts of the amino acid sequence from two peptides  flanking the majority of the peptide sequence identified were used to generate degenerate primers.
degeneracy was limited by taking into account codon usage bias for r. catesbeiana and other identified type i keratin cdna sequences.
two primers with  <dig> fold degeneracy each generated a single  <dig> bp pcr product from r. catesbeiana tail cdna.
based on this sequence two gene specific primers  were designed to perform 5'- and 3'-rapid amplification of cdna ends .
two overlapping clones were obtained from the 5'- and 3'-race containing the entire open reading frame of this keratin gene .
the cloned sequence was  <dig> bp long, with a  <dig> bp 3'-untranslated region, and a polyadenylation signal, aataaa, at  <dig> nucleotides upstream of the poly tract.
the deduced amino acid sequence coded for a  <dig> amino acid protein  and matched exactly all of the observed peptides from the ms analysis indicating that the correct corresponding cdna sequence was cloned.
blastp  <cit>  analysis and clustalw  <cit>  alignment with this  <dig> amino acid sequence revealed the highest identity and similarity  with the x. laevis type i keratin  <dig> kda protein  .
about a dozen keratin proteins have been identified for x. laevis  <cit> .
in r. catesbeiana only four keratin proteins have been defined so far.
these include the adult and larval keratins, rak and rlk, respectively, and a keratin k <dig> and inner-ear cytokeratin  <cit> .
rak is the only acidic type i keratin while the remaining three are basic-neutral type ii keratins.
the sequence we identified is 73% identical  to rak  and 32–33% identical to the type ii keratins: rlk, rana keratin k <dig> and inner-ear cytokeratin .
our sequence also showed 67% identity  to the human type i keratin  <dig>  protein .
based upon this evidence and that presented below, the isolated sequence constitutes a novel rana type i keratin which we will refer to as rlk i. the previous type ii larval rlk will be referred to as rlk ii.
keratins are expressed by epithelial cells where they impart a mechanical function.
in recent years, they have also been shown to be posttranslationally modified during cell stress, apoptosis, and cell signaling  <cit> .
keratins have been extensively studied in anurans during skin differentiation and metamorphosis.
the skin in r. catesbeiana transitions from a larval type into a pre-adult type, and finally into an adult type with the onset of metamorphosis  <cit> .
these changes are associated with the differentiation and apoptosis of specific epidermal cells and changes in connective tissue  <cit> .
each of these changes have been associated with alterations in keratin type expression in specific cells  <cit> .
rlk ii expression was reduced in tadpole tail and body skin with the onset of metamorphosis while rak expression increased  <cit> .
these changes are precociously induced by t <dig> and similar changes have been observed in x. laevis with its corresponding larval and adult keratin genes  <cit> .
rlk i had the highest homology to the x. laevis type i keratin  <dig> kda which is expressed during embryonic and larval stages, reduced during metamorphosis, and expressed at very low levels in the adult skin  <cit> .
in order to determine the transcript levels of rlk i, we performed quantitative real-time polymerase chain reaction  analysis on tail samples from premetamorphic r. catesbeiana tadpoles exposed to t <dig> and during normal tadpole development.
exposure of premetamorphic tadpoles to t <dig> significantly reduced the steady-state levels of the rlk i transcript by  <dig>  and  <dig>  fold at  <dig> and  <dig> h, respectively, relative to time-matched controls .
the same trend was observed during natural metamorphosis.
the steady-state level of the keratin transcript remained unchanged from premetamorphosis , through prometamorphosis  and then decreased by  <dig>  fold upon reaching metamorphic climax  when th levels are maximal and the tail begins to regress   <cit> .
keratins range in size from  <dig> to  <dig> kda.
the keratin spot corresponding to rlk i runs at ~ <dig> kda and all of the peptides that we detected in the ms analysis from the protein spot mapped to the n-terminal end of the complete cloned sequence .
immunoblot analyses of the  <dig> hour microsomal fraction using a pan-cytokeratin antibody revealed the appearance of a similarly migrating fragment in the t <dig> sample with a concomitant reduction of protein intensity at around  <dig> kda compared to the control sample .
interestingly, the identified peptides lie just upstream of what is known as a consensus caspase cleavage site  identified in type i human keratins  <cit>  suggesting that our observed protein spot is a caspase cleavage product of rlk i .
no caspase cleavage of any keratins has previously been reported in anurans during metamorphosis.
however, a number of effector caspases, such as caspase  <dig> and  <dig>  increase in expression and activity during metamorphosis in the tail  <cit> .
caspase  <dig> is the most markedly up-regulated in the tail.
it is expressed in larval skin epidermal cells that undergo th-induced cell autonomous death and is known to act on type i keratins in humans  <cit> .
rlk i was also highly similar to human k <dig> protein.
this type i keratin and the related k <dig> protein form heterodimers with the type ii keratin k <dig> in simple-type epithelia and are a prevalent and extensively-studied group of keratins  <cit> .
both k <dig> and k <dig> are known caspase  <dig> substrates  <cit> .
type i keratin caspase cleavage occurs very early in apoptosis before the detection of dna fragmentation.
type ii keratins are not caspase substrates since they lack the caspase cleavage sequence  <cit> .
in addition, the onset of apoptosis is associated with rapid phosphorylation of both type i and type ii keratins on their head and tail domains.
for k <dig>  ser <dig> phosphorylation controls its caspase cleavage and ser <dig> phosphorylation regulates binding to 14-3- <dig> protein  <cit> .
from our results it is possible that our keratin fragment is also phosphorylated.
like k <dig>  it contains ser residues at positions  <dig> and  <dig>  also, the phosphoprotein stain revealed an increased phosphoprotein spot in the nuclear fraction positioned just slightly towards the more acidic end of the gel relative to the rlk i fragment.
this phosphoprotein was increased at  <dig> and  <dig> h by  <dig>  and  <dig>  fold respectively which matches the increase of the total-protein keratin spot .
although this phosphoprotein could not be identified by ms, its more acidic position adjacent to the rlk i fragment suggests it could be a phosphorylated form of the same fragment.
the reasons for keratin cleavage and phosphorylation are not well understood and are speculated to function as phosphate sinks, for filament re-organization during apoptosis, or mechanisms that protect cells from apoptotic damage allowing a graded sequence of events.
in human cancer therapy, the appearance of the caspase cleavage products of k <dig> and k <dig> in patient serum are used as indicators of cancer prognosis and apoptotic death of tumor cells undergoing chemotherapy  <cit> .
the ease with which this fragment was detected in our immunoblot using a pan-cytokeratin-specific antibody suggests that it could be used as a protein marker for the induction of a th response in tadpoles which may be perturbed upon exposure to disruptors of th action.
the very early appearance of this fragment during precocious tail metamorphosis before the detection of any overt apoptotic events  <cit>  is very intriguing.
it is currently unclear whether the appearance of this keratin fragment is a product of initiation of the apoptotic event or is required for tail regression to occur.
phosphorylation changes in γ-interferon-inducible lysosomal thiol reductase
three phosphoprotein spots located at  <dig> kda and pi ~ <dig> increased in abundance due to t <dig> at  <dig> h in the mitochondrial and microsomal protein fractions .
these spots formed a train indicating that this could be a single protein with different posttranslational modifications.
the phosphoprotein spots increased ~ <dig> fold, while the only corresponding protein spot that could be detected on the total-protein gels did not change in abundance .
this indicates that t <dig> caused a change in the posttranslational state of the protein while its expression level remained the same.
ms analysis of the protein spot from the microsomal and mitochondrial, control and treatment samples indicated that this is the same protein spot .
esi-qqtof ms/ms analysis of the tryptic fragments of the protein spot provided two high quality peptide sequences .
a homology search of the protein database with the two sequences gave the highest match to the xenopus tropicalis γ-interferon-inducible protein  <dig>  , more commonly known as gamma-interferon-inducible lysosomal thiol reductase   <cit> .
this protein also matched the observed molecular weight and pi point.
ms analysis of the only detectable protein spot s <dig> .
1observed peptide masses resulting from the tryptic digestion of the protein spot s <dig>  reported as singly charged.
2peptide sequence information deduced from ms/ms spectra of the corresponding peptides from esi-qqtof analysis.
the masses of isoleucine are indistinguishable from leucine in ms and therefore l can be i and vice versa.
3percent confidence for the peptide sequences, as reported by peaks software for the esi-qqtof spectra.
4highest homology match from protein database searching with the observed peptide sequences to x. tropicalis gilt.
indicates the sequence alignment of the observed peptide to the identified protein  as aligned by blastp.
the sequence in between indicates the matching and similar  amino acids between the two sequences.5from blastp alignments.
gilt is the specific lysosomal enzyme responsible for thiol reduction of proteins in the endocytic pathway for antigen presentation  <cit> .
gamma interferon  released by activated t cells, increases antigen presentation on antigen presenting cells  such as macrophages, dendritic cells, and b cells through the induction of major histocompatability complex ii  molecules and related proteins.
in addition, ifnγ increases the constitutive expression of gilt in apcs and even induces gilt expression in non-hematopoietic cells such as fibroblasts, keratinocytes and endothelial cells  <cit> .
gilt is transported in its proform  from the er and golgi complex into the early endosomes of the endocytic pathway where it is combined with mhcii-invariant chain containing vesicles and converted into its mature form  by the removal of n- and c-terminal prosequences in the late endosomes and lysosomes  <cit> .
it is likely that the gilt protein we identified is the mature form of the protein due to its localization in the microsomal and mitochondrial fractions , the size on the 2d gels, and scope of coverage of identified peptides.
further analyses of the phosphoprotein spots using 2d immunoblots and antibodies specific for the three common phospho-amino acids, phosphoserine, phosphothreonine, and phosphotyrosine did not give any indication that the gilt protein is differentially phosphorylated on any of those residues , while the proq diamond phosphoprotein stain reproducibly detected a change in phosphorylation.
as with many lysosomal enzymes, gilt is a glycoprotein with three potential n-linked glycosylation sites.
furthermore, mature gilt contains mannose-6-phosphate  on one or more of the glycan chains  <cit> .
phosphorylation of mannose within the n-linked glycan chain is a signal recognized in the golgi complex by the m6p receptor that targets lysosomal enzymes to the endocytic pathway  <cit> .
therefore, it is possible that the proq diamond phosphoprotein stain is detecting a phosphate in m6p and that our observed increase in phosphorylation of gilt indicates an increased amount of m6p-containing gilt being directed into the endocytic pathway due to t <dig>  in accordance with this idea of increased endocytic pathway activity, the lysosomal endoprotease, cathepsin d, increases in expression due to t <dig> in our itraq experiment .
cathepsin d, with other lysosomal cathepsin proteases  also converts the proform of gilt into its mature form by cleaving off the n- and c-terminal prosequences  <cit> .
a gilt knock-out study in mice, showed that gilt is required for the presentation of disulfide-containing antigens and the resulting t cell activation  <cit> .
these ideas, and the additional observation of increased immunoglobulin  heavy chains from our itraq experiment , indicate that t <dig> must be stimulating antigen presentation and possibly in turn activating t cell and finally ig-producing/possessing b cell lymphocytes.
additional changes observed in the 2d gel analysis
within the region of the keratin spot, additional proteins and phosphoproteins were increased in the nuclear fraction .
a protein spot that could not be identified increased by  <dig>  and  <dig>  fold at  <dig> and  <dig> h, respectively .
and two unidentified phosphoproteins were increased only at  <dig> h by ~ <dig> fold .
a protein spot located at  <dig> kda and pi ~ <dig>  in the microsomal fraction was increased by  <dig>  fold at  <dig> h upon t <dig> exposure .
amino acid sequence was obtained with high confidence for three peptides from this spot but no significant homology match could be made .
differential expression analysis using itraq
2d gel analysis has the advantage of identifying specific protein isoforms and posttranslational modifications of whole proteins.
this whole-protein analysis approach is limited by the inability to observe hydrophobic proteins and proteins with extremes in size or pi  <cit> .
in contrast, ms analysis techniques circumvent this limit by analyzing peptides derived from trypsin-cleaved protein samples.
however ms peptide analysis has limitations in distinguishing between protein isoforms that potentially share identical peptides  <cit> .
consequently, 2d gel analysis and ms analysis techniques are complementary methods that can be combined to study differential protein expression.
therefore, to increase the number of observed altered proteins, changes in protein expression in the r. catesbeiana tadpole tail fin were additionally assessed using the novel ms technique, itraq  <cit> .
the itraq labeling reagents are four unique chemical tags  that label peptides on primary amines allowing for the quantitation of relative protein abundance in four samples simultaneously during a single analysis .
each sample is labeled with a different tag.
the tags are isobaric, meaning that identical peptides from four different samples will be observed in the ms analysis as a single peptide.
peptide fragmentation and tandem-ms  analysis of that peptide then allows peptide sequence information to be acquired leading to a protein inference.
in addition, different reporter ions are generated from the tags after peptide fragmentation at  <dig>   <dig>   <dig> and  <dig> m/z in the ms/ms spectra, representing each of the four samples, indicating the proteins' relative abundance.
relative abundance is obtained by measuring the area underneath the reporter peaks and is reported as a ratio between the samples.
in our case, the data is reported as the average ratio of the t <dig> treatment versus the control from two duplicate experiments.
a ratio above  <dig> indicates the fold increase in expression due to t <dig> exposure, while a ratio below  <dig> indicates a reciprocal fold decrease in expression.
traditionally, itraq data analysis is performed automatically with software, which greatly reduces the analysis by the end-user.
the success of obtaining valid data in this manner, however, is first dependent on the protein identification obtained using software, such as mascot, where an identification is reliant on a perfect match of ion peaks in a ms/ms spectrum to an existing protein in the protein sequence database  <cit> .
this method works well for the analysis of organisms with extensive entries in gene and protein databases but results in many unidentified spectra when less well-defined species, such as r. catesbeiana, are analyzed.
additionally, when a high quality partial ms/ms spectrum is obtained  software limitations do not permit identification when using ion peaks database searching.
to remedy these problems, we first analyzed the raw itraq data using excel spreadsheets to find all the ms/ms spectra that showed significant changes in expression due to the t <dig> treatment.
the resulting ms/ms spectra were de novo sequenced manually and/or using additional software   <cit> .
the resulting peptide sequences were then used to query protein databases using blastp.
this resulted in substantially higher identification of proteins due to homologous matches or identical matches originally missed by mascot.
to increase the proteome coverage and quantitation accuracy, the itraq samples were analyzed three times on an esi-qqtof mass spectrometer   <cit> .
as shown in table  <dig>  many of the peptides that showed changes in abundance due to t <dig> treatment overlapped between the runs.
the mass spectrometer collected  <dig> to  <dig> unique ms/ms spectra per run.
out of those, ~ <dig> % of the spectra with high quality itraq reporter tags represented peptides that changed according to our criteria of  <dig>  fold or higher.
in total,  <dig> unique spectra showed an increase or decrease of  <dig>  fold or higher due to the t <dig> treatment.
successful de novo peptide sequence information was obtained for  <dig>  of those spectra.
nineteen  of those amino acid sequences were identified with homology protein database searching.
only three of the  <dig> peptides were identified using the standard database matching method that requires a perfect match to a protein database using mascot software.
these  <dig> peptides represent at least  <dig> different proteins,  <dig> of which increased and  <dig> of which decreased in abundance due to t <dig> exposure.
the itraq samples were analyzed three times on an esi-qqtof mass spectrometer.
1indicates number of unique spectra shared between the replicate runs.
2total number of unique spectra recorded per ms analysis run.
these overlap by unknown amount: n/a.
3number of spectra that change by ≥  <dig>  fold due to t <dig>  have good quality reporter tags, and a ratio of the two controls between  <dig>  and  <dig> .
4number of spectra for which amino acid sequence could be obtained through manual sequencing.
5number of spectra for which a protein inference was made with blastp.
6indicates how many spectra were identified using the standard itraq analysis with mascot software.
7number of different proteins identified from the resulting peptides and their change in abundance.
the same itraq samples were also analyzed using a maldi-tof-tof mass spectrometer  to obtain additional proteome coverage because the peptide ionization characteristics are different from the esi-qqtof.
due to software limitations no de novo sequencing of raw ms/ms spectra was performed and the data was analyzed using the standard method of database searching using the observed ms/ms spectra ion peaks using mascot.
the maldi-tof-tof analysis identified  <dig> peptides with over 95% confidence.
these peptides represented  <dig> unique proteins, of which 50% were identified through a single peptide.
four proteins showed altered abundance ≥  <dig>  fold due to the t <dig> treatment.
one of these proteins had an increased abundance while three had a decreased abundance.
details of the itraq results are presented in table  <dig>  interestingly, some of these proteins have established functional connections that may be important during tail regression.
in addition, some of the proteins have connections to those identified in the previous 2d gel analyses.
the identified proteins are involved in apoptotic events, modulation of the extracellular matrix, immune system, metabolism, mechanical function, and oxygen transport and are discussed in detail below.
ltpatv+t
vtfelvyee+lk
flsfpqtk
flsfpqtk
y phfdltpgsadln
vist dls fflr
v+llppsp
s p++ +dgt+tvk
lnv+ dw sgk
ftctvsh+dlpap
q vee+gg lik
+dgt+tvk
aywqir
ayvtvlgdimgta++nldr
gnttsmnfk
tgpagapgqdgrpgppgppgar
ppgpsgek
nicyvithged
nahslylagvfr
digadwvilghser
1name of protein for the highest scoring match that resulted with the observed peptide sequence using blastp  and the ncbi protein database  searching all metazoans.
the ncbi accession number and the species name for the identified protein are shown.
2fold change ratios greater then  <dig>  indicate a fold increase, and those below  <dig>  indicate the reciprocal of fold decrease.
the number is the average fold change of two replicate experiments, which consisted of two treatment samples and two control samples.
3observed mass of the peptide in the ms analysis, modified with the itraq reagent, reported as singly charged.
4amino acid sequence determined for the observed peptide by manual de novo or automatic de novo by peaks software sequencing, or a mascot software match.
indicates that only a partial peptide sequence could be determined.
the masses of isoleucine are indistinguishable from leucine in ms and therefore l can be i and vice versa.
5percent confidence for the peptide sequence as reported by the peaks or mascot  software.
indicates that the peptide was identified in the maldi-tof-tof analysis of itraq samples.
"manual" indicates that the peptide sequence was obtained manually and no percent confidence can be reported.
6indicates the sequence alignment of the observed peptide  to the identified protein  as aligned by blastp.
the sequence in between indicates the matching and similar  amino acids between the two sequences.
7from blastp alignments.
immune system components
t <dig> induces the activation of the immune system as is evident with the increased maturation of gilt as revealed in the 2d analysis.
in addition, this is apparent from an increase in immunoglobulin chains and cathepsin d and is possibly related to the decrease of a coatomer protein.
six peptides that increased  <dig> – <dig>  fold matched a type of immunoglobulin heavy chain .
the peptides matched sequences from x. laevis as well as from other species.
the peptides were derived from constant and variable regions of the heavy chain, and one matched the igm heavy chain isotype.
the immune system plays a major role during the metamorphic process.
macrophages increase in the regressing tadpole tail in number and phagocytic activity removing apoptotic bodies of dying muscle cells  <cit> .
larval lymphocyte populations rise in the growing tadpole, decrease sharply at climax of metamorphosis, and expand again with adult lymphocytes  <cit> .
the exchange in lymphocyte populations appears to be required for the tolerance of new adult antigens and for the removal of larval tissues that are seen as 'non-self' in the metamorphosing animal.
x. laevis tail cells possess larval antigens that stimulate the immune system of a syngeneic adult or a metamorphosing animal  <cit> .
izutsu and coworkers have shown that a  <dig> kda larval antigen expressed specifically by tail epidermal cells only during metamorphosis causes increased proliferation of newly arising adult-type t lymphocytes, eventually leading to the destruction of tail fin tissue by cytotoxic t lymphocytes  and natural killer cells   <cit> .
collaboration between t- and b-cell types must occur for nk cell activation and may explain our observation of increased immunoglobulin heavy chains which can only come from b cell activation.
a reported peptide sequence from the  <dig> kda antigen is homologous to keratin α, and points at the possibility that the increased keratin fragment observed in the 2d gel analyses may be a larval antigen produced to direct the involvement of the immune system.
a peptide that increased by  <dig>  fold was identified in the maldi-tof-tof analysis corresponding to a putative xenopus cathepsin d protein .
cathepsin d is a lysosomal aspartyl protease that has been previously implicated in tadpole tail regression  <cit> .
the increase in cathepsin d may be related to gilt maturation inside the lysosomes and a resulting increase in antigen presentation as discussed above.
a subunit γ protein of the coatomer protein complex  corresponding to the x. laevis mgc <dig> protein decreased by  <dig>  fold in the maldi-tof-tof analysis .
protein transport between the er and golgi compartment is mediated by these non-clathrin-coated vesicular coat protein complexes  <cit> .
copi is composed of seven unique subunits and coats the vesicles on the cytoplasmic side as they are transported from one organelle to another.
copi is mainly associated with retrograde transport of vesicles from the golgi back to the er to retrieve escaped er-resident proteins and vesicle machinery.
the γ subunit binds double lysine motifs in the transmembrane proteins targeted for return to the er.
interestingly, gilt and other lysosomal zymogens have dilysine motifs which are used as cleavage sites during enzyme maturation.
there have not been any previous associations of copi with th or metamorphosis, but it is possible that t <dig> causes a specific change in vesicle trafficking to accomodate tail regression.
this change may contribute to an increase in mature gilt in the lysosomes as is observed on the 2ds of the microsomal and mitochondrial fractions.
extracellular matrix components and modifier proteins
changes in the extracellular matrix  of the regressing tadpole tail have been well documented  <cit> .
four proteins involved in ecm structure and modification were identified by itraq .
the plasma proteinase inhibitor, α-2-macroglobulin , was decreased  <dig>  fold upon t <dig> treatment .
a2m is synthesized by several cell types such as hepatocytes, lung fibroblasts and monocyte-macrophages, and functions to trap and facilitate clearing of proteases such as trypsin, thrombin and collagenases  <cit> .
tissue-specific a2m synthesis is independent of plasma a2m and serves a compartmentalized function such as the regulation of tissue proteinases such as matrix metalloproteinases.
since there is an increase in matrix metalloprotease activity in the tadpole tail in th-induced and natural metamorphosis  <cit> , the observed decrease in the a2m levels could be a result of an organized downregulation to allow for ecm remodeling or a2m and metalloprotease covalent linkage and subsequent removal and degradation of the proteins.
recently, lin et al.
observed a t3-dependent decrease in a2m transcripts in a human hepatocellular carcinoma cell line  <cit> .
a peptide matching inter-α inhibitor h <dig> protein increased by  <dig>  fold .
the h <dig> polypeptide is a less described component of the trimeric plasma serine protease inhibitor, inter-α inhibitor , more commonly composed of the h <dig>  h <dig> and bikunin polypeptides  <cit> .
iai proteins are linked to inflammation processes and ecm structure.
serine protease inhibitor activity of bikunin regulates plasmin which is a known activator of matrix metalloproteases  <cit> .
the h <dig> polypeptide is also a serum marker for the acute-phase response in infected animals and humans resulting from the nonspecific immune response  <cit>  and may be an additional link to our observation of the immune system activation.
additionally, the h polypeptides stabilize and protect the ecm through covalent links to ecm components and possibly through collagen binding domains  <cit> .
α  <dig> type i collagen  chain) was identified by two peptides that decreased by  <dig> – <dig>  fold .
type i collagen is the most abundant member of the collagen family and is the main constituent of the ecm  <cit> .
it is a major substrate of matrix metalloproteinase  <dig>  which is upregulated in r. catesbeiana tail and back skin during natural and t3-induced metamorphosis  <cit> .
collagen  and α 2) gene expression also decreased under these conditions in the tail and intestine  <cit> .
it is possible that the α  <dig> collagen gene is negatively regulated by a tre-like site as in rat cardiac fibroblasts  <cit> .
here we show that this change also occurs at the protein level.
the last peptide associated with ecm remodeling proteins corresponds with cortactin .
a  <dig>  fold decrease in this src homology  <dig>  domain-containing protein suggests altered tyrosine kinase signaling of actin cytoskeleton rearrangements  <cit> .
cortactin is a filamentous-actin binding protein that stabilizes actin networks and promotes actin polymerization.
since cortactin interacts with various proteins involved in actin polymerization, endocytosis  and cell-cell interactions, modification of any or all of these processes could contribute to tail regression.
cell metabolism proteins
two proteins involved in cell metabolic homeostasis were altered in abundance by t <dig> treatment .
the first peptide was increased by  <dig>  fold matched with a sterol regulatory element binding protein- <dig> .
this membrane-bound transcription factor regulates lipid homeostasis through the control of genes encoding key enzymes important in cholesterol synthesis  <cit> .
the srebp transcription factors have not been implicated in anuran metamorphosis before, however, they are involved with th in the mammalian context  <cit> .
recently shin and osborne  <cit>  observed that t <dig> regulates both srebp- <dig> mrna and nuclear protein levels, as well as srebp-2-controlled genes such as the low density lipoprotein  receptor in mice.
the srebp- <dig> promoter contains at least one tr-binding site and is activated directly by tr in a ligand-dependent manner  <cit> .
it is therefore possible that the increased level of srebp- <dig> observed in our experiments may be a direct response to t <dig>  this remains to be determined.
the second peptide was reduced by  <dig>  fold and corresponds to triose phosphate isomerase .
this enzyme is involved in glycolysis  <cit>  and its decrease is compatible with a shift from glycolysis to oxidative phosphorylation observed during metamorphosis  <cit> .
in addition, two proteins involved in oxygen transport were increased upon t <dig> treatment.
three peptides corresponding to larval hemoglobin α-iii chain increased by  <dig>  fold .
this protein is only found in tadpole red blood cells  and not adult rbcs  <cit> .
in r. catesbeiana there is a change in erythropoiesis sites and hemoglobin types during tadpole growth and metamorphosis that is influenced by ths  <cit> .
young tadpoles produce hemoglobin components i and ii in the kidneys, older tadpoles produce components iii and iv in the liver, and after metamorphosis, only adult hemoglobins are produced in the bone marrow.
this sequential change produces hemoglobins with lower oxygen affinities.
our observation of an increase in hemoglobin α-iii is indicative of an accelerated progression of hemoglobin switching due to t <dig> exposure.
consistent with a switch in hemoglobin is an  <dig>  fold increase in a peptide matching biliverdin reductase b .
this enzyme is important in hemoglobin metabolism and is predominantly found in human fetal liver  <cit> .
there, it produces bilirubin ixβ from biliverdin derived from heme.
bilirubin ixβ is the major heme catabolite during human fetal development  <cit> .
in adult human liver biliverdin reductase a  is the major form and it produces bilirubin ixα.
the switch in this heme degradation pathway seems to be related to the switch in hemoglobins from fetal to adult forms.
mechanical function and calcium modulated proteins
the abundance of peptides from muscle α-actin, myosin heavy chain  and myosin light chain  was reduced by exactly the same amount .
these proteins are the fundamental components of the molecular machinery used in muscle contraction.
the peptide for muscle α-actin is distinguished from the β and γ forms of cytoplasmic actins by a t residue instead of v in the hptll sequence within this peptide.
the peptide sequence matches the r. catesbeiana cardiac α-actin  <dig> sequence perfectly as well as other muscle α actins in other species.
the mhc peptide matches the r. catesbeiana skeletal muscle mhc- <dig> and is distinguishable from the other isoforms, mhcs - <dig>   <dig>   <dig>  and  <dig>  by three residues in this species  <cit> .
the mlc peptide matches the x. laevis mlc- <dig>  as well as mlc- <dig> proteins from other species.
an embryonic/larval α- <dig> skeletal-actin gene has been found in xenopus borealis  <cit> .
this gene was expressed in the skeletal muscle of embryos and tadpoles, but not in adults.
however, the corresponding gene in x. laevis is highly expressed in both adult muscle and at earlier stages.
the human skeletal muscle α-actin gene is a direct th response gene in cardiocytes  <cit> .
the cardiac α-actin gene of rats is also inducible with t <dig> in heart myocytes  <cit> .
five skeletal muscle mhc isoforms have been cloned in r. catesbeiana  <cit> .
no specific larval forms have been found and all five isoforms are expressed in the tail and limb muscle of both tadpoles and adults.
in the tadpole tail muscle, t <dig> increases the mrna for mhcs - <dig>   <dig>   <dig>  and  <dig> by two to three-fold within  <dig> h  <cit> .
however, in contrast, the rate of total mhc protein synthesis was reduced by 40% at  <dig> h  <cit>  consistent with our observations for mhc- <dig>  in fast twitch muscle fibers, mhc- <dig> is associated with mlc- <dig>  <cit> .
mlcs are ca2+-signaling dependent regulators of muscle contraction and have been investigated in x. laevis and r. pipiens but their responses to th are not well understood  <cit> .
our data suggest that mhc and mlc expression are co-regulated.
a peptide that was decreased by  <dig>  fold matched the p26olf protein of r. catesbeiana  the p26olf protein is a s100-like ca2+-binding protein with the very unique feature of having two s100-like regions  <cit> .
thus far p26olf has only been found in the anurans r. catesbeiana, x. tropicalis and x. laevis, and appears not to have any homologues in other vertebrates.
immunohistochemical studies on r. catesbeiana frogs showed that p26olf primarily localizes in the cilia of olfactory and lung respiratory epithelia.
p26olf may be involved in olfactory signal transduction and a related protein, s100/a <dig> , is a key mediator of high ca2+-induced growth arrest and differentiation in human keratinocytes  <cit> .
in addition, makino et al.
<cit>  have shown that introducing an n-terminal peptide of s100/a <dig> into human carcinoma cell lines induced apoptosis.
thus, it may be possible that p26olf is involved in the control of apoptotic events in the tadpole tail.
CONCLUSIONS
genomic studies on tadpole tail regression have revealed a number of modulated transcripts  <cit> .
in comparison, our extensive proteomic analyses showed relatively few changes within the first  <dig> h of t <dig> treatment.
this provides support for the hypothesis that changes in the transcriptome are the major target for the establishment of the tail regression program.
however, this does not imply that the few changes in the proteome during this time are not important, as these changes may support the transcriptome transition.
for example, the tyrosine kinase inhibitor, genistein, prevents t3-dependent tail regression likely through inhibition of protein kinase c activity and trα phosphorylation  <cit> .
our proteomic analyses may be limited in their sensitivity to reveal phosphorylation of low-abundance signaling phosphoproteins since no enrichment was performed.
nevertheless our data suggest that rlk i could be posttranslationally modified due to a t <dig> signal most likely through the early action of caspases thereby indicating an early apoptotic event.
our findings with gilt maturation, ig heavy chains and cathepsin d increases show that the involvement of the immune system in the regressing tadpole tail may be greater than what has been generally considered.
furthermore, a large proportion of the transcripts identified by genomic studies encode transcription factors  <cit>  whose protein products are most likely below the detection limits of our 2d page and itraq analyses.
an improved fractionation strategy for the itraq analysis may reveal these.
the present study provides important insights into the molecular mechanism of t3-dependent tadpole tail regression during postembryonic development which includes novel components at the proteome level.

 thyroid hormones are important signaling molecules in vertebrates that regulate homeostasis, growth, and development. one developmental process that is dependent upon the presence of th is amphibian metamorphosis. during metamorphosis the larval, aquatic, herbivorous tadpole transforms into a terrestrial, carnivorous juvenile frog. this event requires drastic changes in essentially every organ and tissue of the tadpole and includes: the resorption of larval organs and tissues, remodeling of larval organs into juvenile form, and de novo development of organs and tissues. metamorphosis is completely controlled through the control of serum th levels. the thyroid gland mainly produces the thyroid hormone,,3',5'-tetraiodothyronine, which is converted into the biologically more active form,,3'-triiodothyronine, in the peripheral tissues. progression through natural metamorphosis is dependent upon a tightly-controlled rise and fall in th levels. premetamorphic tadpoles are functionally athyroid with no measurable levels of ths. th levels gradually increase during prometamorphosis and reach maximal levels at metamorphic climax. at this stage, overt remodelling of the tadpole rapidly ensues. premetamorphic tadpoles can be induced to undergo precocious metamorphosis by exposure to th. the best understood mechanism of th action involves th binding to nuclear thyroid hormone receptors which regulate gene expression. th binding to trs can either activate or repress transcription of responsive genes through recruitment of coactivators and corepressors, respectively. differential nuclear tr levels and intracellular t levels controlled by deiodinase activity and th-binding proteins, contribute to tissue-specific responses. however, the response to th also depends on the existing complement of other proteins that can influence cell fate. however, the molecular mechanisms are poorly understood. most th-responsive genes are up-regulated and these have been most commonly studied particularly in the tail. the genetic program required for tail regression is established between and h of th exposure at a "commitment point" after which removal of th or exposure to transcription or protein synthesis inhibitors cannot prevent regression. studies based on pcr subtractive hybridization methods and cdna gene arrays have identified a number of possible genes involved in this process. however, the relationship of most of these genomic findings to changes in the proteome has yet to be identified and there is growing evidence for non-classical th action through phosphorylation signaling pathways. proteomic scale studies on th-dependent tail regression have been scarce. ray et al. identified several 35s-methionine labelled proteins from rana catesbeiana tail fin that change during natural and precocious metamorphosis using two-dimensional gel analysis. kobayashi et al. used 2d gel electrophoresis to analyze changes in protein expression in the back and tail skin of xenopus laevis during metamorphosis. from the 2d protein spot patterns they could classify the back skin into larval or adult type and observe the transition. attempts were made in these studies to identify the altered protein spots. this, however, involved identifying the spots based on position, comigration or immunological detection methods. using 2d gel separation and mass spectrometry for protein identification, we were able to identify proteins whose expression was altered in the x. laevis tadpole tail during th-induced metamorphosis. regardless of the approach, the lack of any sample fractionation led to the identification of only abundant proteins. in this work we identified novel changes in the proteome and phosphoproteome associated with the induction of t3-dependent tail regression of rana catesbeiana tadpoles. proteins that changed in abundance were detected using two-dimensional gel electrophoresis and isobaric tags for relative and absolute quantitation methods. alterations in phosphorylation were revealed using a phosphoprotein-specific stain. mass spectrometry analyses were then used for protein identification. proteome coverage was enhanced through the use of cell and protein fractionation to reveal several proteins that are altered within h of t exposure.  fractionation of the tail fin proteome and 2d gel analyses the subcellular fractionation method was optimized to provide highly enriched fractions of cytosolic, mitochondrial, nuclear and microsomal samples from the r. catesbeiana tail fin. the goals of the method were to limit sample complexity with effective cell fractionation with minimal cross-contamination and reasonable yields. the fractions also had to be salt and buffer-compatible with subsequent proteomic analysis. we developed two separate procedures based on differential centrifugation to generate nuclear and cytosolic/mitochondrial/microsomal fractions. the nuclear extraction procedure was optimized to minimize nuclear clumping, increase nuclei stability, and minimize cytoskeletal, cytoplasmic/organelle and dna contamination. the cytosolic/mitochondrial/microsomal extraction procedure was developed to increase the disruption of tail fin cell membrane and increase mitochondrial stability and purity. the mitochondria-enriched fraction was obtained with a × g centrifugation and it likely also contained lysosomes, peroxisomes, golgi and endoplasmic reticulum. centrifugation at × g removed the vesicles of the plasma membrane, endosomes, golgi and er into the microsomal pellet leaving the cytosolic supernatant with its soluble molecules. nuclear integrity was monitored by microscopy and fractionation efficiency was determined using immunoblot analysis for subcellular markers. an immunoblot for cytochrome c shows substantial enrichment for that organelle in the mitochondrial fraction while an immunoblot for the nuclear markers, lamins b and b shows enrichment of nuclei in the nuclear fraction. the cytosolic fraction is a complex mixture of many proteins and was therefore further fractionated using anion-exchange high performance liquid chromatography. a step-gradient anion-exchange hplc procedure was developed that used ammonium bicarbonate as a volatile buffer in place of commonly used salt and non-volatile buffer to provide salt-free fractions after lyophilization to render the samples compatible with the subsequent 2d gel analysis. the cytosolic fraction was thus further fractionated into five fractions: mm, mm, mm, mm and m ammonium bicarbonate with each fraction yielding roughly equal amounts of protein as shown by sds-page. proteins within each of the resulting fractions were then separated by 2d polyacrylamide gel electrophoresis which separates proteins based on their molecular weight and isoelectric point. therefore, the entire fractionation protocol divided the tail fin proteome over eight 2d gels: nuclear, mitochondrial, microsomal and five cytosolic fractions. this fractionation method increases the ability to observe expression changes in low abundance proteins and provides information on subcellular localization of proteins which cannot be achieved by examining a whole cell homogenate on a single 2d gel. from our results, it is evident that each of the fractions shows a distinctive pattern of spots with many unique spots per fraction analyzed. there is some overlap with the more abundant protein spots between the neighboring fractions of the hplc separation and between the microsomal and mitochondrial fractions, which probably share many cellular membrane compartments. phosphoproteins were detected on the 2d gels with a phosphoprotein-specific fluorescent stain which detects phosphorylation on ser, thr and tyr residues. total proteins and phosphoproteins were detected in the same 2d gel allowing for easy identification of phosphoprotein location and subsequent isolation for ms analysis. the above methods were used to analyze the proteome and phosphoproteome of the premetamorphic r. catesbeiana tail fin undergoing precocious metamorphosis at and h induced with nm t a minimum of three independent replicates allowed for the verification of changes in protein and phosphoprotein expression and ms analysis was used for protein identification. identification of a unique r. catesbeiana keratin fragment a prominent protein spot at ~ kda and pi ~ was increased upon t treatment on the 2d gels of several fractions. it was observed in the mm cytoplasmic fraction as well as in the microsomal, mitochondrial and nuclear fractions. this protein spot was increased by 2– fold as early as h, but was more intensely expressed at h. the greatest increase was observed in the microsomal fraction. the protein spots from each of the fractions were separately analyzed by mass spectrometry proving that each fraction represented the same protein. a combination of electrospray-ionization quadrupole time-of-flight and matrix-assisted laser desorption ionization tof-tof tandem-ms analyses allowed for peptide sequence information to be obtained for different peptides from this protein. protein database searches with these peptides gave the highest homology match to the x. laevis type i keratin kda protein also known as x. laevis keratin b [ncbi: 1304283b) from the xk gene family. 1observed peptide masses resulting from the tryptic digestion of the protein spot, reported as singly charged. 2peptide sequence information deduced from ms/ms spectra of the corresponding peptides from esi-qqtof analysis. the masses of isoleucine are indistinguishable from leucine in ms and therefore l can be i and vice versa. 3indicates which peptides were additionally observed with maldi-tof-tof analysis. 4percent confidence for the peptide sequences as reported by peaks software for the esi-qqtof spectra and by mascot for maldi-tof-tof data. 5highest homology match from protein database searching with the observed peptide sequences to x. laevis type i keratin kd using spider software. bold lettering indicates differences between the observed and database sequences. parts of the amino acid sequence from two peptides flanking the majority of the peptide sequence identified were used to generate degenerate primers. degeneracy was limited by taking into account codon usage bias for r. catesbeiana and other identified type i keratin cdna sequences. two primers with fold degeneracy each generated a single bp pcr product from r. catesbeiana tail cdna. based on this sequence two gene specific primers were designed to perform 5'- and 3'-rapid amplification of cdna ends. two overlapping clones were obtained from the 5'- and 3'-race containing the entire open reading frame of this keratin gene. the cloned sequence was bp long, with a bp 3'-untranslated region, and a polyadenylation signal, aataaa, at nucleotides upstream of the poly tract. the deduced amino acid sequence coded for a amino acid protein and matched exactly all of the observed peptides from the ms analysis indicating that the correct corresponding cdna sequence was cloned. blastp analysis and clustalw alignment with this amino acid sequence revealed the highest identity and similarity with the x. laevis type i keratin kda protein. about a dozen keratin proteins have been identified for x. laevis. in r. catesbeiana only four keratin proteins have been defined so far. these include the adult and larval keratins, rak and rlk, respectively, and a keratin k and inner-ear cytokeratin. rak is the only acidic type i keratin while the remaining three are basic-neutral type ii keratins. the sequence we identified is 73% identical to rak and 32–33% identical to the type ii keratins: rlk, rana keratin k and inner-ear cytokeratin. our sequence also showed 67% identity to the human type i keratin protein. based upon this evidence and that presented below, the isolated sequence constitutes a novel rana type i keratin which we will refer to as rlk i. the previous type ii larval rlk will be referred to as rlk ii. keratins are expressed by epithelial cells where they impart a mechanical function. in recent years, they have also been shown to be posttranslationally modified during cell stress, apoptosis, and cell signaling. keratins have been extensively studied in anurans during skin differentiation and metamorphosis. the skin in r. catesbeiana transitions from a larval type into a pre-adult type, and finally into an adult type with the onset of metamorphosis. these changes are associated with the differentiation and apoptosis of specific epidermal cells and changes in connective tissue. each of these changes have been associated with alterations in keratin type expression in specific cells. rlk ii expression was reduced in tadpole tail and body skin with the onset of metamorphosis while rak expression increased. these changes are precociously induced by t and similar changes have been observed in x. laevis with its corresponding larval and adult keratin genes. rlk i had the highest homology to the x. laevis type i keratin kda which is expressed during embryonic and larval stages, reduced during metamorphosis, and expressed at very low levels in the adult skin. in order to determine the transcript levels of rlk i, we performed quantitative real-time polymerase chain reaction analysis on tail samples from premetamorphic r. catesbeiana tadpoles exposed to t and during normal tadpole development. exposure of premetamorphic tadpoles to t significantly reduced the steady-state levels of the rlk i transcript by and fold at and h, respectively, relative to time-matched controls. the same trend was observed during natural metamorphosis. the steady-state level of the keratin transcript remained unchanged from premetamorphosis, through prometamorphosis and then decreased by fold upon reaching metamorphic climax when th levels are maximal and the tail begins to regress. keratins range in size from to kda. the keratin spot corresponding to rlk i runs at ~ kda and all of the peptides that we detected in the ms analysis from the protein spot mapped to the n-terminal end of the complete cloned sequence. immunoblot analyses of the hour microsomal fraction using a pan-cytokeratin antibody revealed the appearance of a similarly migrating fragment in the t sample with a concomitant reduction of protein intensity at around kda compared to the control sample. interestingly, the identified peptides lie just upstream of what is known as a consensus caspase cleavage site identified in type i human keratins suggesting that our observed protein spot is a caspase cleavage product of rlk i. no caspase cleavage of any keratins has previously been reported in anurans during metamorphosis. however, a number of effector caspases, such as caspase and increase in expression and activity during metamorphosis in the tail. caspase is the most markedly up-regulated in the tail. it is expressed in larval skin epidermal cells that undergo th-induced cell autonomous death and is known to act on type i keratins in humans. rlk i was also highly similar to human k protein. this type i keratin and the related k protein form heterodimers with the type ii keratin k in simple-type epithelia and are a prevalent and extensively-studied group of keratins. both k and k are known caspase substrates. type i keratin caspase cleavage occurs very early in apoptosis before the detection of dna fragmentation. type ii keratins are not caspase substrates since they lack the caspase cleavage sequence. in addition, the onset of apoptosis is associated with rapid phosphorylation of both type i and type ii keratins on their head and tail domains. for k ser phosphorylation controls its caspase cleavage and ser phosphorylation regulates binding to 14-3- protein. from our results it is possible that our keratin fragment is also phosphorylated. like k it contains ser residues at positions and also, the phosphoprotein stain revealed an increased phosphoprotein spot in the nuclear fraction positioned just slightly towards the more acidic end of the gel relative to the rlk i fragment. this phosphoprotein was increased at and h by and fold respectively which matches the increase of the total-protein keratin spot. although this phosphoprotein could not be identified by ms, its more acidic position adjacent to the rlk i fragment suggests it could be a phosphorylated form of the same fragment. the reasons for keratin cleavage and phosphorylation are not well understood and are speculated to function as phosphate sinks, for filament re-organization during apoptosis, or mechanisms that protect cells from apoptotic damage allowing a graded sequence of events. in human cancer therapy, the appearance of the caspase cleavage products of k and k in patient serum are used as indicators of cancer prognosis and apoptotic death of tumor cells undergoing chemotherapy. the ease with which this fragment was detected in our immunoblot using a pan-cytokeratin-specific antibody suggests that it could be used as a protein marker for the induction of a th response in tadpoles which may be perturbed upon exposure to disruptors of th action. the very early appearance of this fragment during precocious tail metamorphosis before the detection of any overt apoptotic events is very intriguing. it is currently unclear whether the appearance of this keratin fragment is a product of initiation of the apoptotic event or is required for tail regression to occur. phosphorylation changes in γ-interferon-inducible lysosomal thiol reductase three phosphoprotein spots located at kda and pi ~ increased in abundance due to t at h in the mitochondrial and microsomal protein fractions. these spots formed a train indicating that this could be a single protein with different posttranslational modifications. the phosphoprotein spots increased ~ fold, while the only corresponding protein spot that could be detected on the total-protein gels did not change in abundance. this indicates that t caused a change in the posttranslational state of the protein while its expression level remained the same. ms analysis of the protein spot from the microsomal and mitochondrial, control and treatment samples indicated that this is the same protein spot. esi-qqtof ms/ms analysis of the tryptic fragments of the protein spot provided two high quality peptide sequences. a homology search of the protein database with the two sequences gave the highest match to the xenopus tropicalis γ-interferon-inducible protein, more commonly known as gamma-interferon-inducible lysosomal thiol reductase. this protein also matched the observed molecular weight and pi point. ms analysis of the only detectable protein spot s. 1observed peptide masses resulting from the tryptic digestion of the protein spot s reported as singly charged. 2peptide sequence information deduced from ms/ms spectra of the corresponding peptides from esi-qqtof analysis. the masses of isoleucine are indistinguishable from leucine in ms and therefore l can be i and vice versa. 3percent confidence for the peptide sequences, as reported by peaks software for the esi-qqtof spectra. 4highest homology match from protein database searching with the observed peptide sequences to x. tropicalis gilt. indicates the sequence alignment of the observed peptide to the identified protein as aligned by blastp. the sequence in between indicates the matching and similar amino acids between the two sequences.5from blastp alignments. gilt is the specific lysosomal enzyme responsible for thiol reduction of proteins in the endocytic pathway for antigen presentation. gamma interferon released by activated t cells, increases antigen presentation on antigen presenting cells such as macrophages, dendritic cells, and b cells through the induction of major histocompatability complex ii molecules and related proteins. in addition, ifnγ increases the constitutive expression of gilt in apcs and even induces gilt expression in non-hematopoietic cells such as fibroblasts, keratinocytes and endothelial cells. gilt is transported in its proform from the er and golgi complex into the early endosomes of the endocytic pathway where it is combined with mhcii-invariant chain containing vesicles and converted into its mature form by the removal of n- and c-terminal prosequences in the late endosomes and lysosomes. it is likely that the gilt protein we identified is the mature form of the protein due to its localization in the microsomal and mitochondrial fractions, the size on the 2d gels, and scope of coverage of identified peptides. further analyses of the phosphoprotein spots using 2d immunoblots and antibodies specific for the three common phospho-amino acids, phosphoserine, phosphothreonine, and phosphotyrosine did not give any indication that the gilt protein is differentially phosphorylated on any of those residues, while the proq diamond phosphoprotein stain reproducibly detected a change in phosphorylation. as with many lysosomal enzymes, gilt is a glycoprotein with three potential n-linked glycosylation sites. furthermore, mature gilt contains mannose-6-phosphate on one or more of the glycan chains. phosphorylation of mannose within the n-linked glycan chain is a signal recognized in the golgi complex by the m6p receptor that targets lysosomal enzymes to the endocytic pathway. therefore, it is possible that the proq diamond phosphoprotein stain is detecting a phosphate in m6p and that our observed increase in phosphorylation of gilt indicates an increased amount of m6p-containing gilt being directed into the endocytic pathway due to t in accordance with this idea of increased endocytic pathway activity, the lysosomal endoprotease, cathepsin d, increases in expression due to t in our itraq experiment. cathepsin d, with other lysosomal cathepsin proteases also converts the proform of gilt into its mature form by cleaving off the n- and c-terminal prosequences. a gilt knock-out study in mice, showed that gilt is required for the presentation of disulfide-containing antigens and the resulting t cell activation. these ideas, and the additional observation of increased immunoglobulin heavy chains from our itraq experiment, indicate that t must be stimulating antigen presentation and possibly in turn activating t cell and finally ig-producing/possessing b cell lymphocytes. additional changes observed in the 2d gel analysis within the region of the keratin spot, additional proteins and phosphoproteins were increased in the nuclear fraction. a protein spot that could not be identified increased by and fold at and h, respectively. and two unidentified phosphoproteins were increased only at h by ~ fold. a protein spot located at kda and pi ~ in the microsomal fraction was increased by fold at h upon t exposure. amino acid sequence was obtained with high confidence for three peptides from this spot but no significant homology match could be made. differential expression analysis using itraq 2d gel analysis has the advantage of identifying specific protein isoforms and posttranslational modifications of whole proteins. this whole-protein analysis approach is limited by the inability to observe hydrophobic proteins and proteins with extremes in size or pi. in contrast, ms analysis techniques circumvent this limit by analyzing peptides derived from trypsin-cleaved protein samples. however ms peptide analysis has limitations in distinguishing between protein isoforms that potentially share identical peptides. consequently, 2d gel analysis and ms analysis techniques are complementary methods that can be combined to study differential protein expression. therefore, to increase the number of observed altered proteins, changes in protein expression in the r. catesbeiana tadpole tail fin were additionally assessed using the novel ms technique, itraq. the itraq labeling reagents are four unique chemical tags that label peptides on primary amines allowing for the quantitation of relative protein abundance in four samples simultaneously during a single analysis. each sample is labeled with a different tag. the tags are isobaric, meaning that identical peptides from four different samples will be observed in the ms analysis as a single peptide. peptide fragmentation and tandem-ms analysis of that peptide then allows peptide sequence information to be acquired leading to a protein inference. in addition, different reporter ions are generated from the tags after peptide fragmentation at and m/z in the ms/ms spectra, representing each of the four samples, indicating the proteins' relative abundance. relative abundance is obtained by measuring the area underneath the reporter peaks and is reported as a ratio between the samples. in our case, the data is reported as the average ratio of the t treatment versus the control from two duplicate experiments. a ratio above indicates the fold increase in expression due to t exposure, while a ratio below indicates a reciprocal fold decrease in expression. traditionally, itraq data analysis is performed automatically with software, which greatly reduces the analysis by the end-user. the success of obtaining valid data in this manner, however, is first dependent on the protein identification obtained using software, such as mascot, where an identification is reliant on a perfect match of ion peaks in a ms/ms spectrum to an existing protein in the protein sequence database. this method works well for the analysis of organisms with extensive entries in gene and protein databases but results in many unidentified spectra when less well-defined species, such as r. catesbeiana, are analyzed. additionally, when a high quality partial ms/ms spectrum is obtained software limitations do not permit identification when using ion peaks database searching. to remedy these problems, we first analyzed the raw itraq data using excel spreadsheets to find all the ms/ms spectra that showed significant changes in expression due to the t treatment. the resulting ms/ms spectra were de novo sequenced manually and/or using additional software. the resulting peptide sequences were then used to query protein databases using blastp. this resulted in substantially higher identification of proteins due to homologous matches or identical matches originally missed by mascot. to increase the proteome coverage and quantitation accuracy, the itraq samples were analyzed three times on an esi-qqtof mass spectrometer. as shown in table many of the peptides that showed changes in abundance due to t treatment overlapped between the runs. the mass spectrometer collected to unique ms/ms spectra per run. out of those, ~ % of the spectra with high quality itraq reporter tags represented peptides that changed according to our criteria of fold or higher. in total, unique spectra showed an increase or decrease of fold or higher due to the t treatment. successful de novo peptide sequence information was obtained for of those spectra. nineteen of those amino acid sequences were identified with homology protein database searching. only three of the peptides were identified using the standard database matching method that requires a perfect match to a protein database using mascot software. these peptides represent at least different proteins, of which increased and of which decreased in abundance due to t exposure. the itraq samples were analyzed three times on an esi-qqtof mass spectrometer. 1indicates number of unique spectra shared between the replicate runs. 2total number of unique spectra recorded per ms analysis run. these overlap by unknown amount: n/a. 3number of spectra that change by ≥ fold due to t have good quality reporter tags, and a ratio of the two controls between and. 4number of spectra for which amino acid sequence could be obtained through manual sequencing. 5number of spectra for which a protein inference was made with blastp. 6indicates how many spectra were identified using the standard itraq analysis with mascot software. 7number of different proteins identified from the resulting peptides and their change in abundance. the same itraq samples were also analyzed using a maldi-tof-tof mass spectrometer to obtain additional proteome coverage because the peptide ionization characteristics are different from the esi-qqtof. due to software limitations no de novo sequencing of raw ms/ms spectra was performed and the data was analyzed using the standard method of database searching using the observed ms/ms spectra ion peaks using mascot. the maldi-tof-tof analysis identified peptides with over 95% confidence. these peptides represented unique proteins, of which 50% were identified through a single peptide. four proteins showed altered abundance ≥ fold due to the t treatment. one of these proteins had an increased abundance while three had a decreased abundance. details of the itraq results are presented in table interestingly, some of these proteins have established functional connections that may be important during tail regression. in addition, some of the proteins have connections to those identified in the previous 2d gel analyses. the identified proteins are involved in apoptotic events, modulation of the extracellular matrix, immune system, metabolism, mechanical function, and oxygen transport and are discussed in detail below. ltpatv+t vtfelvyee+lk flsfpqtk flsfpqtk y phfdltpgsadln vist dls fflr v+llppsp s p++ +dgt+tvk lnv+ dw sgk ftctvsh+dlpap q vee+gg lik +dgt+tvk aywqir ayvtvlgdimgta++nldr gnttsmnfk tgpagapgqdgrpgppgppgar ppgpsgek nicyvithged nahslylagvfr digadwvilghser 1name of protein for the highest scoring match that resulted with the observed peptide sequence using blastp and the ncbi protein database searching all metazoans. the ncbi accession number and the species name for the identified protein are shown. 2fold change ratios greater then indicate a fold increase, and those below indicate the reciprocal of fold decrease. the number is the average fold change of two replicate experiments, which consisted of two treatment samples and two control samples. 3observed mass of the peptide in the ms analysis, modified with the itraq reagent, reported as singly charged. 4amino acid sequence determined for the observed peptide by manual de novo or automatic de novo by peaks software sequencing, or a mascot software match. indicates that only a partial peptide sequence could be determined. the masses of isoleucine are indistinguishable from leucine in ms and therefore l can be i and vice versa. 5percent confidence for the peptide sequence as reported by the peaks or mascot software. indicates that the peptide was identified in the maldi-tof-tof analysis of itraq samples. "manual" indicates that the peptide sequence was obtained manually and no percent confidence can be reported. 6indicates the sequence alignment of the observed peptide to the identified protein as aligned by blastp. the sequence in between indicates the matching and similar amino acids between the two sequences. 7from blastp alignments. immune system components t induces the activation of the immune system as is evident with the increased maturation of gilt as revealed in the 2d analysis. in addition, this is apparent from an increase in immunoglobulin chains and cathepsin d and is possibly related to the decrease of a coatomer protein. six peptides that increased – fold matched a type of immunoglobulin heavy chain. the peptides matched sequences from x. laevis as well as from other species. the peptides were derived from constant and variable regions of the heavy chain, and one matched the igm heavy chain isotype. the immune system plays a major role during the metamorphic process. macrophages increase in the regressing tadpole tail in number and phagocytic activity removing apoptotic bodies of dying muscle cells. larval lymphocyte populations rise in the growing tadpole, decrease sharply at climax of metamorphosis, and expand again with adult lymphocytes. the exchange in lymphocyte populations appears to be required for the tolerance of new adult antigens and for the removal of larval tissues that are seen as'non-self' in the metamorphosing animal. x. laevis tail cells possess larval antigens that stimulate the immune system of a syngeneic adult or a metamorphosing animal. izutsu and coworkers have shown that a kda larval antigen expressed specifically by tail epidermal cells only during metamorphosis causes increased proliferation of newly arising adult-type t lymphocytes, eventually leading to the destruction of tail fin tissue by cytotoxic t lymphocytes and natural killer cells. collaboration between t- and b-cell types must occur for nk cell activation and may explain our observation of increased immunoglobulin heavy chains which can only come from b cell activation. a reported peptide sequence from the kda antigen is homologous to keratin α, and points at the possibility that the increased keratin fragment observed in the 2d gel analyses may be a larval antigen produced to direct the involvement of the immune system. a peptide that increased by fold was identified in the maldi-tof-tof analysis corresponding to a putative xenopus cathepsin d protein. cathepsin d is a lysosomal aspartyl protease that has been previously implicated in tadpole tail regression. the increase in cathepsin d may be related to gilt maturation inside the lysosomes and a resulting increase in antigen presentation as discussed above. a subunit γ protein of the coatomer protein complex corresponding to the x. laevis mgc protein decreased by fold in the maldi-tof-tof analysis. protein transport between the er and golgi compartment is mediated by these non-clathrin-coated vesicular coat protein complexes. copi is composed of seven unique subunits and coats the vesicles on the cytoplasmic side as they are transported from one organelle to another. copi is mainly associated with retrograde transport of vesicles from the golgi back to the er to retrieve escaped er-resident proteins and vesicle machinery. the γ subunit binds double lysine motifs in the transmembrane proteins targeted for return to the er. interestingly, gilt and other lysosomal zymogens have dilysine motifs which are used as cleavage sites during enzyme maturation. there have not been any previous associations of copi with th or metamorphosis, but it is possible that t causes a specific change in vesicle trafficking to accomodate tail regression. this change may contribute to an increase in mature gilt in the lysosomes as is observed on the 2ds of the microsomal and mitochondrial fractions. extracellular matrix components and modifier proteins changes in the extracellular matrix of the regressing tadpole tail have been well documented. four proteins involved in ecm structure and modification were identified by itraq. the plasma proteinase inhibitor, α-2-macroglobulin, was decreased fold upon t treatment. a2m is synthesized by several cell types such as hepatocytes, lung fibroblasts and monocyte-macrophages, and functions to trap and facilitate clearing of proteases such as trypsin, thrombin and collagenases. tissue-specific a2m synthesis is independent of plasma a2m and serves a compartmentalized function such as the regulation of tissue proteinases such as matrix metalloproteinases. since there is an increase in matrix metalloprotease activity in the tadpole tail in th-induced and natural metamorphosis, the observed decrease in the a2m levels could be a result of an organized downregulation to allow for ecm remodeling or a2m and metalloprotease covalent linkage and subsequent removal and degradation of the proteins. recently, lin et al. observed a t3-dependent decrease in a2m transcripts in a human hepatocellular carcinoma cell line. a peptide matching inter-α inhibitor h protein increased by fold. the h polypeptide is a less described component of the trimeric plasma serine protease inhibitor, inter-α inhibitor, more commonly composed of the h h and bikunin polypeptides. iai proteins are linked to inflammation processes and ecm structure. serine protease inhibitor activity of bikunin regulates plasmin which is a known activator of matrix metalloproteases. the h polypeptide is also a serum marker for the acute-phase response in infected animals and humans resulting from the nonspecific immune response and may be an additional link to our observation of the immune system activation. additionally, the h polypeptides stabilize and protect the ecm through covalent links to ecm components and possibly through collagen binding domains. α type i collagen chain) was identified by two peptides that decreased by – fold. type i collagen is the most abundant member of the collagen family and is the main constituent of the ecm. it is a major substrate of matrix metalloproteinase which is upregulated in r. catesbeiana tail and back skin during natural and t3-induced metamorphosis. collagen and α 2) gene expression also decreased under these conditions in the tail and intestine. it is possible that the α collagen gene is negatively regulated by a tre-like site as in rat cardiac fibroblasts. here we show that this change also occurs at the protein level. the last peptide associated with ecm remodeling proteins corresponds with cortactin. a fold decrease in this src homology domain-containing protein suggests altered tyrosine kinase signaling of actin cytoskeleton rearrangements. cortactin is a filamentous-actin binding protein that stabilizes actin networks and promotes actin polymerization. since cortactin interacts with various proteins involved in actin polymerization, endocytosis and cell-cell interactions, modification of any or all of these processes could contribute to tail regression. cell metabolism proteins two proteins involved in cell metabolic homeostasis were altered in abundance by t treatment. the first peptide was increased by fold matched with a sterol regulatory element binding protein-. this membrane-bound transcription factor regulates lipid homeostasis through the control of genes encoding key enzymes important in cholesterol synthesis. the srebp transcription factors have not been implicated in anuran metamorphosis before, however, they are involved with th in the mammalian context. recently shin and osborne observed that t regulates both srebp- mrna and nuclear protein levels, as well as srebp-2-controlled genes such as the low density lipoprotein receptor in mice. the srebp- promoter contains at least one tr-binding site and is activated directly by tr in a ligand-dependent manner. it is therefore possible that the increased level of srebp- observed in our experiments may be a direct response to t this remains to be determined. the second peptide was reduced by fold and corresponds to triose phosphate isomerase. this enzyme is involved in glycolysis and its decrease is compatible with a shift from glycolysis to oxidative phosphorylation observed during metamorphosis. in addition, two proteins involved in oxygen transport were increased upon t treatment. three peptides corresponding to larval hemoglobin α-iii chain increased by fold. this protein is only found in tadpole red blood cells and not adult rbcs. in r. catesbeiana there is a change in erythropoiesis sites and hemoglobin types during tadpole growth and metamorphosis that is influenced by ths. young tadpoles produce hemoglobin components i and ii in the kidneys, older tadpoles produce components iii and iv in the liver, and after metamorphosis, only adult hemoglobins are produced in the bone marrow. this sequential change produces hemoglobins with lower oxygen affinities. our observation of an increase in hemoglobin α-iii is indicative of an accelerated progression of hemoglobin switching due to t exposure. consistent with a switch in hemoglobin is an fold increase in a peptide matching biliverdin reductase b. this enzyme is important in hemoglobin metabolism and is predominantly found in human fetal liver. there, it produces bilirubin ixβ from biliverdin derived from heme. bilirubin ixβ is the major heme catabolite during human fetal development. in adult human liver biliverdin reductase a is the major form and it produces bilirubin ixα. the switch in this heme degradation pathway seems to be related to the switch in hemoglobins from fetal to adult forms. mechanical function and calcium modulated proteins the abundance of peptides from muscle α-actin, myosin heavy chain and myosin light chain was reduced by exactly the same amount. these proteins are the fundamental components of the molecular machinery used in muscle contraction. the peptide for muscle α-actin is distinguished from the β and γ forms of cytoplasmic actins by a t residue instead of v in the hptll sequence within this peptide. the peptide sequence matches the r. catesbeiana cardiac α-actin sequence perfectly as well as other muscle α actins in other species. the mhc peptide matches the r. catesbeiana skeletal muscle mhc- and is distinguishable from the other isoforms, mhcs - and by three residues in this species. the mlc peptide matches the x. laevis mlc- as well as mlc- proteins from other species. an embryonic/larval α- skeletal-actin gene has been found in xenopus borealis. this gene was expressed in the skeletal muscle of embryos and tadpoles, but not in adults. however, the corresponding gene in x. laevis is highly expressed in both adult muscle and at earlier stages. the human skeletal muscle α-actin gene is a direct th response gene in cardiocytes. the cardiac α-actin gene of rats is also inducible with t in heart myocytes. five skeletal muscle mhc isoforms have been cloned in r. catesbeiana. no specific larval forms have been found and all five isoforms are expressed in the tail and limb muscle of both tadpoles and adults. in the tadpole tail muscle, t increases the mrna for mhcs - and by two to three-fold within h. however, in contrast, the rate of total mhc protein synthesis was reduced by 40% at h consistent with our observations for mhc- in fast twitch muscle fibers, mhc- is associated with mlc-. mlcs are ca2+-signaling dependent regulators of muscle contraction and have been investigated in x. laevis and r. pipiens but their responses to th are not well understood. our data suggest that mhc and mlc expression are co-regulated. a peptide that was decreased by fold matched the p26olf protein of r. catesbeiana the p26olf protein is a s100-like ca2+-binding protein with the very unique feature of having two s100-like regions. thus far p26olf has only been found in the anurans r. catesbeiana, x. tropicalis and x. laevis, and appears not to have any homologues in other vertebrates. immunohistochemical studies on r. catesbeiana frogs showed that p26olf primarily localizes in the cilia of olfactory and lung respiratory epithelia. p26olf may be involved in olfactory signal transduction and a related protein, s100/a, is a key mediator of high ca2+-induced growth arrest and differentiation in human keratinocytes. in addition, makino et al. have shown that introducing an n-terminal peptide of s100/a into human carcinoma cell lines induced apoptosis. thus, it may be possible that p26olf is involved in the control of apoptotic events in the tadpole tail.  genomic studies on tadpole tail regression have revealed a number of modulated transcripts. in comparison, our extensive proteomic analyses showed relatively few changes within the first h of t treatment. this provides support for the hypothesis that changes in the transcriptome are the major target for the establishment of the tail regression program. however, this does not imply that the few changes in the proteome during this time are not important, as these changes may support the transcriptome transition. for example, the tyrosine kinase inhibitor, genistein, prevents t3-dependent tail regression likely through inhibition of protein kinase c activity and trα phosphorylation. our proteomic analyses may be limited in their sensitivity to reveal phosphorylation of low-abundance signaling phosphoproteins since no enrichment was performed. nevertheless our data suggest that rlk i could be posttranslationally modified due to a t signal most likely through the early action of caspases thereby indicating an early apoptotic event. our findings with gilt maturation, ig heavy chains and cathepsin d increases show that the involvement of the immune system in the regressing tadpole tail may be greater than what has been generally considered. furthermore, a large proportion of the transcripts identified by genomic studies encode transcription factors whose protein products are most likely below the detection limits of our 2d page and itraq analyses. an improved fractionation strategy for the itraq analysis may reveal these. the present study provides important insights into the molecular mechanism of t3-dependent tadpole tail regression during postembryonic development which includes novel components at the proteome level. 
these proteins are involved in apoptosis, extracellular matrix structure, immune system, metabolism, mechanical function, and oxygen transport. altered proteins were identified using mass spectrometry. furthermore, gilt, a protein involved in the immune system, is changed in phosphorylation state which is linked to its activation. we identified and cloned a novel rana larval type i keratin, rlk i, which may be a target for caspase-mediated proteolysis upon exposure to t in addition, the rlk i transcript is reduced during t3-induced and natural metamorphosis which is consistent with a larval keratin. to this end, we developed a cell and protein fractionation method combined with two-dimensional gel electrophoresis and phosphoprotein-specific staining. thyroid hormones are vital in the maintenance of homeostasis and in the control of development. in addition, there is increasing evidence that changes in the phosphoproteome influence th action. one postembryonic developmental process that is principally regulated by ths is amphibian metamorphosis. we have demonstrated the ability to derive proteomics-based information from a model species for postembryonic development for which no genome information is currently available. using a complementary ms technique for the analysis of differentially-expressed proteins, isobaric tags for relative and absolute quantitation revealed additional proteins whose levels were altered upon t treatment. the present study identifies proteins whose levels and/or phosphorylation states are altered within h of the induction of tadpole tail regression prior to overt remodeling of the tail.
 thyroid hormones are vital in the maintenance of homeostasis and in the control of development. one postembryonic developmental process that is principally regulated by ths is amphibian metamorphosis. this process has been intensively studied at the genomic level yet very little information at the proteomic level exists. in addition, there is increasing evidence that changes in the phosphoproteome influence th action.  here we identify components of the proteome and phosphoproteome in the tail fin that changed within h of exposure of premetamorphic rana catesbeiana tadpoles to nm,3'-triiodothyronine. to this end, we developed a cell and protein fractionation method combined with two-dimensional gel electrophoresis and phosphoprotein-specific staining. altered proteins were identified using mass spectrometry. we identified and cloned a novel rana larval type i keratin, rlk i, which may be a target for caspase-mediated proteolysis upon exposure to t in addition, the rlk i transcript is reduced during t3-induced and natural metamorphosis which is consistent with a larval keratin. furthermore, gilt, a protein involved in the immune system, is changed in phosphorylation state which is linked to its activation. using a complementary ms technique for the analysis of differentially-expressed proteins, isobaric tags for relative and absolute quantitation revealed additional proteins whose levels were altered upon t treatment. the success of identifying proteins whose levels changed upon t treatment with itraq was enhanced through de novo sequencing of ms data and homology database searching. these proteins are involved in apoptosis, extracellular matrix structure, immune system, metabolism, mechanical function, and oxygen transport.  we have demonstrated the ability to derive proteomics-based information from a model species for postembryonic development for which no genome information is currently available. the present study identifies proteins whose levels and/or phosphorylation states are altered within h of the induction of tadpole tail regression prior to overt remodeling of the tail. in particular, we have identified a novel keratin that is a target for t3-mediated changes in the tail that can serve as an indicator of early response to this hormone. 