BACKGROUND
many aspects of modern biological research are dependent on accurate identification of the protein-coding genes in each genome, as well as the nature of the mature functional protein products, a process commonly referred to as genome annotation.
with the exponential increase in the number of sequenced prokaryotic genomes afforded by advances in genome sequencing technologies over the last decade, present day prokaryotic genome annotation is essentially an automated high-throughput process that relies heavily on de novo gene prediction programs  <cit> .
while de novo gene prediction programs have significantly improved for prokaryotic genomes considerable challenges remain  <cit> , such as determining the precise start and stop site of a gene, accurately predicting short genes, and determining a stop codon that represents an alternative amino acid rather than a true stop site.
as efforts to sequence more branches of the tree of life expand, the level of accuracy for current gene prediction programs trained on proteobacteria datasets will markedly decrease, leading to an increase in incorrect predictions of protein-coding genes  <cit> .
compounding the issue is the lack of experimental evidence in support of predicted protein-coding regions for the overwhelming majority of annotated genomes.
where available, experimental evidence is typically based on expressed rna sequences, such as from microarray or rna seq experiments.
however, these genome-centric analyses do not independently and unequivocally determine whether a predicted protein-coding gene is translated into a protein or importantly provide any reliable information on post-translational processing.
bottom-up proteomics offers the ability to directly measure peptides arising from expressed proteins representing the current best option for independently and unambiguously identifying at least an important subset of the protein-coding genes in a genome and can be used to experimentally validate gene annotations  <cit> .
in a bottom up approach, proteins within a complex mixture are typically digested with a protease, after which the resulting peptides are separated by chromatographic methods and then analyzed using tandem mass spectrometry   <cit> .
each ms/ms spectrum is a measure of fragment masses, ideally from a single peptide sequence of ~ 6- <dig> amino acids.
this set of mass values is analogous to a 'fingerprint' that identifies the peptide.
interpretation of ms/ms peptide spectra is accomplished 1) by using algorithms such as x!tandem  <cit> , sequest  <cit> , or mascot  <cit>  to compare measured masses against a set of theoretical masses of possible protein sequences or 2) less commonly, by de novo analysis, which does not depend on any prior knowledge of the possible sequences  <cit> .
similar to searching ms/ms spectra against a set of predicted protein sequences, it is also possible  to identify the protein-coding genes in a genome by searching ms/ms spectra against a six-frame translation of the genomic dna sequence, thereby precluding the inherent biases derived from gene prediction methods.
we note that scaling up to an exponentially larger database, as a result of six-frame translation of a genomic dna sequence, makes searches slower by potentially orders of magnitude.
additionally, it also results in increased possibility for false-positive identifications as the false discovery rate scales with the increasing database size, greatly decreasing sensitivity; twin challenges that can only be feasibly met with dedicated/sophisticated computing resources precluding routine use at present.
bottom-up proteomics also provides an avenue for obtaining biologically relevant information about post-translational modifications.
even for relatively simple biological systems such as prokaryotes, post-translational modification  events are increasingly being recognized as important, but are poorly characterized with regard to sites or function.
while ptm information gained from genome-scale ms/ms datasets stands to benefit biological understanding of bacterial organisms, significant technological challenges hamper the routine inclusion of this valuable information in primary annotations.
this situation is exemplified by only a single report of genome-scale ms/ms datasets being used to comprehensively annotate post-translational modifications events in a genome sequence  <cit> .
however, the low resolution of the ms/ms datasets makes assignment of modifications, including crucially the site of modification, less confident and ill suited for routine use.
for example, the  <dig>  dalton mass difference between gln  and lys  cannot be resolved in low resolution ms/ms spectra, which increases the number of possible peptide candidates to be matched and reduces confidence in assignments.
on the other hand, the sequencing precision afforded by high resolution ms/ms spectra can resolve residues with small mass differences like gln and lys, which reduces the search space and thus the computational burden, and increases confidence in assignments.
in this study, we employed a bottom-up proteomics approach supplemented with a recently described de novo sequencing methodology using high resolution and high mass measurement accuracy ms/ms data  <cit>  to accurately uncover the translational landscape and post-translational features of the bacterial pathogen salmonella enterica serovar typhimurium   <dig>  salmonella typhimurium is a leading cause of bacterial gastroenteritis and is widely used as a model to investigate basic genetic mechanisms as well as the interaction between bacterial pathogens and mammalian hosts.
in spite of the clinical and basic science relevance of salmonella typhimurium there has been no comprehensive analysis undertaken to provide experimental support of its in silico-based genome annotation to facilitate systems-level analysis.
our data provides protein-level experimental validation for approximately half of the predicted protein-coding genes in stm  <dig> and suggests revisions to  <dig> genes assigned incorrect translational start sites, including a potential novel alternate start codon.
additionally, we uncovered  <dig> non-annotated genes missed by gene prediction programs, as well as evidence suggesting a role for one of these novel orfs in salmonella pathogenesis.
we also characterized post-translational features in the stm  <dig> genome, including chemical modifications and proteolytic cleavages.
we find that bacteria have a much larger and complex repertoire of chemical modifications than previously thought including several novel modifications.
our in vivo proteolysis data identified more than  <dig> signal peptide and n-terminal methionine cleavage events critical for protein function.
contrary to the overwhelming majority of proteogenomics analyses that utilize proteomics data to improve the quality and completeness of previously annotated genomes, this study represents one of the first to utilize proteomics data "as part of a largely automated, high-throughput annotation process directly at the primary stage of genome annotation"  <cit> .
RESULTS
the genome of salmonella typhimurium  14028s was sequenced as described in the methods section and is composed of two replicons: a main chromosome  and a plasmid  with over 99% sequence homology to the salmonella typhimurium lt <dig> virulence plasmid pslt.
the automated annotation pipeline at the j. craig venter institute  was employed to identify genome features in the raw dna sequence, gather evidence for function of the features, and assign functional annotation.
the main genome characteristics of stm  <dig> are presented in table  <dig>  and the full list of predicted protein-coding genes is provided in additional file  <dig> table s <dig>  for the proteogenomic analysis described in this study we focused on chromosome-encoded genome features.
low resolution liquid chromatography -ms/ms datasets were employed to experimentally validate gene annotations and high resolution lc-ms/ms datasets were employed to accurately annotate post-translational features as described below.
to confirm and correct stm  <dig> annotations and in particular to identify proteins specified by non-annotated genes, we searched low resolution ms/ms spectra from  <dig> individual lc-ms/ms analyses against a six-frame translation of the stm  <dig> genome sequence.
these data comprise stm  <dig> samples from different cell culture conditions designed to mimic various aspects of the non-infectious and infectious environments experienced by salmonella typhimurium.
briefly, these included luria-bertani  logarithmic and lb stationary phases, and two acidic minimal medium  conditions, amm <dig> and amm <dig>  <cit> .
the mass spectrometry datasets employed in this proteogenomic annotation study have been previously described, detailing the annotated salmonella typhimurium proteome response to the above growth conditions  <cit> .
peptides with either one or two tryptic ends were filtered according to the parameters described in the methods section.
as an additional confidence metric we only retained those filter-passing peptides that mapped to unique locations  in the six-frame translation.
peptides mapping to multiple locations  in the six-frame translation introduce a potential source of ambiguity making it difficult to confidently validate individual orfs or correct annotation errors.
a total of  <dig> unique peptides that passed the above criteria were identified with a false discovery rate of < 1%.
of these,  <dig> peptides were assigned as being generated by two tryptic ends and  <dig>  with one tryptic end.
experimental validation of predicted genes at the protein level
for the majority of genes annotated as part of sequencing efforts there is no direct experimental evidence that the gene is translated into a protein.
in cases where experimental evidence is available it is typically based on expressed rna sequences, such as from microarray or rnaseq experiments.
it is clear however that expressed rna sequences cannot independently and unequivocally determine whether a predicted protein-coding gene is translated into a protein.
thus knowing that a gene or part of it in the relevant frame is being made  is useful information.
multiple unique peptides mapping to a single gene provide compelling evidence for the expression of the product encoded by that particular gene.
as such, we matched the  <dig>  identified unique peptides to the  <dig> predicted chromosomal genes in the stm  <dig> genome sequence to validate automated gene predictions at the protein level.
the predicted genes covered most of the observed peptides .
conservatively, a gene product was confirmed expressed at the protein level only when a minimum of two unique peptides mapped to the particular gene.
using this criterion, our proteomics data validated  <dig> of the  <dig> annotated chromosomal orfs in the stm <dig> genome sequence at the protein level , providing experimental evidence for the products of these genes.
figure  <dig> shows an example of a predicted gene whose length is covered by multiple identified peptides providing experimental evidence for its expression at the protein level.
given the limited number of possible tryptic peptides available for protein identification in small orfs, the above criteria would presumably preclude the confident identification of novel small orfs.
the ~44% level of proteome coverage is in line with, and in most published cases exceeds, results obtained in both typical proteomics experiments  <cit>  and previous proteogenomic analyses  <cit> .
typical coverage is in the 30% range for the annotated orfs.
however, multiple factors potentially prevent 100% coverage, including extensive post-transcriptional regulation that makes transcripts rather poor guides for protein expression  <cit> .
here we have used four differing growth conditions including typical laboratory conditions and an infection-like environment.
however, not all possible growth conditions for salmonella typhimurium can be efficiently sampled and it is likely that certain orfs require specific conditions for expression, such as intracellular growth in host cells  <cit> .
in addition, various technical aspects associated with our experiments may have precluded detection, such as incompatibility with buffers for soluble protein extraction.
the two peptide criterion may also negatively impact coverage of small proteins present in low concentrations.
however it is possible that application of additional protein and peptide separation methods could enable improved coverage of low abundance proteins.
refinement of predicted gene structures: start sites
determining the correct start position of a gene remains a challenge for current gene prediction algorithms.
in a recent re-analysis of  <dig> annotated prokaryotic genomes, nielsen and krogh  <cit>  observed that in some genomes up to 60% of the genes may have been annotated with a wrong start codon, especially in gc-rich genomes.
accurate start site predictions better define intergenic spaces that may encode promoters and regulatory binding sites, which are critical elements in studies of transcriptional regulation.
cellular localization signals also are contained in start sites, which makes accurate start site predictions important for accurately determining the localization of proteins within a cell.
peptides that map to genomic regions within  <dig> bp upstream of previously annotated genes and in the same translational reading frame represent evidence supporting extensions of their predicted start sites.
we note that this approach has been described in detail in a number of recent publications  <cit> .
additionally peptides that span  the start site of previously annotated genes also represent evidence supporting extensions of their predicted start sites.
to this end, we examined the identified peptides for experimental evidence supporting extension of start sites in each of the predicted genes, and where possible proposed a new start codon.
<dig> peptides spanned the start site of previously annotated genes, and  <dig> peptides mapped to genomic regions within 200bp upstream of previously annotated genes.
overall,  <dig> of the  <dig>  peptides mapped to genomic regions that served as experimental evidence supporting extension of start sites.
we restricted our correction of start sites to predicted genes confirmed at the protein level , which yielded a final candidate list of  <dig> peptides that correspond to  <dig> genes requiring n-terminal extension/start site correction .
new start sites were largely defined by the first methionine amino acid and/or start codon encountered upstream of peptides mapped to the genomic region within  <dig> bp upstream of their previously annotated start sites.
figures  <dig> and  <dig> highlight examples depicting the utility of proteomics data for start site correction.
orf <dig> is a member of the universal stress protein  family of proteins with biological roles in motility, adhesion and coping with stress  <cit> .
in our proteomics data we observed several peptides upstream of orf <dig> in the same reading frame and within  <dig> bp of the predicted start site, i.e., evidence supporting extension of the predicted start site .
furthermore, three of these peptides span the previously predicted start site, providing additional evidence in support of refining the start site.
orf <dig> is the nadh-dependent enoyl reductase from the type ii bacterial fatty acid biosynthesis pathway , as inferred from the primary in silico genome annotation, a validated but currently underexploited target for drug discovery  <cit> .
similarly, we observed several peptides that overlap this orf including one peptide that spanned the currently predicted gene start .
the accurate experimental delineation of the structure of this clinically relevant gene represents an important step in facilitating ongoing drug development efforts.
while beyond the scope of this study, we remark that protein expression and purification coupled with edman degradation represents a useful orthogonal avenue to validate at the protein level the corrected assignments proposed.
in prokaryotes the initiation of translation is typically mediated by the start codon atg.
in addition gtg and ttg are also used as alternative start codon.
studies in e. coli have estimated the frequency of start codon usage as atg 83%, gtg 14% and ttg 3%.
however a very small number of studies have also reported ctg, att, ata, and atc codons to function as rare non-canonical translation initiators.
interestingly our proteomics data showed multiple peptides that overlap orf <dig>  including one peptide that spanned the currently predicted gene start, providing evidence in support of extending the start site .
the peptide, giepmaltkaemseylfdklglskr, is immediately adjacent to a stop codon  with another stop codon  a further seven amino acids upstream making the possibility that the peptide results from a stop codon read through unlikely.
considering this, the first amino acid in the peptide sequence glycine  would presumably be the translation initiator.
glycine is coded for by the triplet codon ggg.
however, it is well known that even if an alternative start codon is used as the translation initiator, it gets translated as methionine.
thus we examined the possibility of a frame-shift involving the preceding orf phet, coded in a different frame, as a potential mechanism to explain this observation.
however, the orf phet terminates with the "...aslrd-stop" amino acid sequence conserved across several related salmonella and e. coli strains.
it is clear that further studies beyond the scope of the present work will be required to resolve and or confirm this interesting observation.
the peptide giepmaltkaemseylfdklglskr is defined as fully tryptic, having a native n-terminus and a c-terminal arginine, and was observed in three independent lc-ms/ms analyses with minimum sequest® peptide identification scores across the three independent lc-ms/ms analyses of xcorr ≥  <dig> , Δcn ≥  <dig> , and peptideprophet™ probability ≥  <dig> .
a representative ms/ms spectrum of the peptide giepmaltkaemseylfdklglskr is shown in figure  <dig>  while we do not observe any fragments ions corresponding to proline directed fragmentation, the near complete y ion series and extensive sequence coverage when considering both b and y ion series provides additional confidence to the annotation.
identification of novel genes
the identification of protein-coding genes  in eukaryotic genomes is complicated by the high frequency of alternative splicing in most eukaryotic genes.
additionally the small fraction of protein-coding genes that comprise eukaryotic genomes  make the identification of coding sequences against the ubiquitous background of non-coding sequences difficult  <cit> .
in contrast, the usual absence of introns and the compact nature of prokaryotic genomes make the identification of all possible orfs longer than a chosen threshold in a dna sequence a relatively straightforward computational exercise.
this view, in particular of bacterial genomes, has lead to the assumption that computationally derived coding sequences completely describe the entire coding capacity of a bacterial genome.
however, structural annotation of prokaryotic genome sequences by predicting coding sequences is far from being a trivial matter.
a number of recent studies  <cit>  used proteomics data to identify novel protein-coding genes in prokaryotic genomes that had been missed by de-novo gene finding programs.
peptides that map to genomic regions outside the boundaries of predicted genes are evidence suggestive of the presence of novel genes missed by gene finding programs.
by matching the  <dig>  identified peptides to the  <dig> predicted chromosomal genes in the stm  <dig> genome sequence, we detected  <dig> peptides mapped to regions falling outside the boundaries of known protein coding genes.
we further refined the list of peptides by excluding the  <dig> peptides that mapped to genomic regions within  <dig> bp upstream of predicted genes, i.e., peptides that are indicative of n-terminal extensions , which yielded a final candidate list of  <dig> peptides .
these peptides represent experimental evidence for the presence of novel genes missed by gene finding programs.
using the set of  <dig> intergenic peptides, we defined  <dig> novel genes that had been missed by gene prediction methods.
note that novel genes were confirmed as detected and expressed at the protein level only when a minimum of two peptides mapped to that particular novel gene.
of particular interest was the novel orf c1368_1:795109- <dig> defined by five peptides that map to the genomic region  <dig> to  <dig> where no gene had been predicted .
protein blast analysis  of the genomic region where the five peptides mapped to against the non-redundant protein sequences database revealed a single significant hit to a phage protein in salmonella typhimurium strain d <dig> .
interestingly, examination of our experimental ms/ms data showed that this novel orf c1368_1:795109- <dig> was highly expressed under growth in acidic minimal media  relative to growth under standard laboratory conditions .
growth in amm has been shown to approximate the environment found within the salmonella-containing vacuoles  observed in infected host macrophages, and we among others have shown that this media induces the expression of the salmonella pathogenicity island  <dig> -encoded type-iii secretion system , a system critical for virulence and intramacrophage survival  <cit> .
thus proteins co-regulated with known virulence proteins under such environmental conditions are likely to play a role in salmonella typhimurium pathogenesis.
indeed phage proteins are known to play an important role in salmonella typhimurium pathogenesis further supporting our observation  <cit> .
the full repertoire of factors employed by salmonella typhimurium to establish a successful infection is still unknown, presumably because they remain undetected due to a number of reasons including being missed by annotators; thus the enumeration of all proteins in the salmonella typhimurium  <dig> genome, especially those co-regulated with known virulence factors, as presented above is an important step in understanding the mechanisms of salmonella typhimurium pathogenesis.
assessing the performance of proteogenomic annotation
manual curation has become a luxury reserved for a few organisms and the majority of newly sequenced genomes only receive a single round of computational annotation with no additional manual refinement, therefore the utility of proteogenomics lies in its application at the primary stage of genome annotation to improve the quality and completeness of the automated genome annotation.
a version of the stm <dig> genome annotated using a combination of computational methods and human annotators, i.e.
refined annotation, released while this manuscript was in preparation  <cit>  allowed us to assess the performance of proteogenomic annotation.
a comparison of the new start sites for the  <dig> genes proposed by the proteomics data to the start sites for the same  <dig> genes in the refined stm <dig> genome annotation revealed an overlap of  <dig> genes where start sites matched exactly .
of the remaining  <dig> genes,  <dig> had proteomics-suggested start sites that were downstream of those determined by refined annotation.
while the proteomics data suggest a shorter orf than indicated in the refined annotation, homology analysis  supports the refined annotation.
note that deeper proteome coverage may improve matches to the refined annotation.
the remaining three genes had proteomics-suggested start sites that were upstream of start sites in the refined annotation, which is tangible experimental evidence that suggests a longer orf than indicated in the refined annotation, i.e., correcting the refined annotation.
in summary  <dig> of  <dig> start site corrections proposed by our proteogenomic analysis in the primary annotation reported in this study were in line with the refined annotation.
the remaining three start site corrections suggested represent corrections to both the automated  and refined annotation  <cit> .
our proteogenomic analysis also identified  <dig> novel genes missed by gene prediction methods.
a comparison of the  <dig> novel genes proposed by proteomics data to the refined stm <dig> genome annotation revealed  <dig> of the  <dig> novel genes were also identified and annotated similarly in the refined annotation .
this result highlights the power of proteomics data to identify relevant novel protein-coding genes.
the three remaining proteomics-identified genes missed by de novo gene finding programs and human annotators represent a correction and improvement to both the current automated  and refined  <cit>  annotations.
annotation of complex post-translational chemical modifications
while a number of groups have used genome-scale ms/ms data to confirm predicted bacterial genes at the protein level, as well as identify new genes and correct gene prediction  <cit> , there is only a single report of genome-scale ms/ms data being used for comprehensive "unrestricted" analysis and annotation of post-translational chemical modifications  in a bacterial system  <cit> .
we note however a number of recent studies focused on characterizing ribosomal protein modifications  <cit> .
as relatively little is known about ptcms in bacteria, even for intensively studied model organisms such as e. coli and salmonella, any ptcm information gained from genome-scale ms/ms data would aid biological understanding of bacterial organisms.
we have recently described a de novo sequencing approach , using high resolution and high mass measurement accuracy ms/ms data, for the accurate discovery of unknown or unexpected ptcms of proteins  <cit> .
here we apply the de novo-ustags approach to analyze  <dig> high resolution lc-ms/ms datasets for ptcms in the stm  <dig> genome.
these datasets represented samples that had been grown in a variety of cell culture conditions.
using the approach illustrated above, we determined mass shifts for ustags in  <dig> ms/ms spectra that represented  <dig> proteins.
we estimate the false discovery rate  of ptcm analysis to be < 1% .
utilizing in-house developed software and a list of  <dig> unimod modifications, including snp substitutions, http://www.unimod.org  we inferred hypothetical explanations for observed mass shifts in ~92% of the spectra in which modifications were detected , several of which had multiple potential chemical explanations .
where applicable observation of "differential" fragments were used to resolve ambiguity of assignments between snps and other modifications of similar mass defect as described below and in the methods and additional file  <dig>  the remaining spectra  in which modifications were detected but where no plausible modification combination of up to two of the selected  <dig> unimod modifications could explain observed mass shift for prefix or suffix sequence represent novel previously un-described ptcms and are listed in additional file  <dig> table s5e.
to obtain lower limit estimates on the number of distinct ptcms, we binned different mass shifts and inferred a total of  <dig> distinct modifications mass-shifts, each with multiple potential chemical explanations according to unimod .
this represents a much larger and complex repertoire of chemical modifications than previously thought existed in bacteria.
note that this only considered mass-shifts for which a potential chemical explanation was found in unimod, thus most likely an underestimate.
nevertheless this estimate represents a much larger number of modification types than can be considered by commonly used, but often restrictive ptcm search algorithms such as sequest, x!tandem, and mascot  <cit> .
the  <dig> modification containing ms/ms spectra with hypothetical explanations for the observed mass shifts were ranked using a peptide identification scoring function  and based on the identification scores we obtained unambiguous explanations, with regards to type, number and site of modification, for observed mass shifts in  <dig> ms/ms spectra .
we also established confident assignment of number and type of modification to explain observed mass shifts in an additional  <dig> ms/ms spectra ; however site of modification could not be unambiguously derived from spectral evidence .
among the modification types observed were those known to result from sample preparation, including carbamylation and carbamidomethyl, as well as those that can occur both in vitro and in vivo, such as methionine oxidation  <cit>  and asparagine deamidation  <cit> .
as no methods are currently available for distinguishing between in vitro and in vivo modifications, to identify ptcms in stm that are biologically relevant we assumed such modifications would be conserved across closely related organisms.
using this approach we report a number of ptcms of biological significance previously unappreciated in stm.
methylation of ribosomal proteins has been suggested to modulate the intra- or intermolecular interactions of the methylated ribosomal proteins or affects their affinity for rna, and, thus, influences various cell processes, including ribosome assembly and translation accuracy  <cit> .
single methylation of the e. coli ribosomal protein l7/ <dig> has been reported  <cit>  and localized to k <dig>  <cit> , while the ribosomal protein l <dig> has been reported as being methylated at q <dig> in e. coli  <cit> .
in the present study we observe methylation of the e. coli ribosomal protein l7/ <dig> homolog in stm <dig> orf <dig> at the same location, k <dig>  and methylation of the ribosomal protein l <dig> ortholog in stm <dig> orf <dig> at the exact same position, q <dig>  suggesting a similar regulatory and/or structural role in stm <dig> as in e. coli .
methylation of the translation elongation factor tu  at position k <dig> has been reported, and suggested as a mechanism for 'fine tuning' of tufb-trna inter-molecular interactions  <cit> .
we observed the same modification at k <dig> of the stm <dig> protein orf <dig>  the homologous position to k <dig> of the e. coli protein, suggesting a similar functional role in stm <dig> as in e. coli .
besides those ptcms with functional inferences from comparative analysis we observed additional salmonella typhimurium ptcms of interest.
for example, figure  <dig> shows the detection of a cyano modification at residue cys <dig> of the salmonella typhimurium protein ribosomal protein s <dig> .
note that the cyano modification is classified in unimod as a post-translational modification and not a chemical derivative/artifact.
this to our knowledge is the first report of an in vivo cyano modification in salmonella.
previous studies in e. coli have reported on the effects of cyanate on the different catalytic activities of carbamyl phosphate synthetase  <cit> .
it will be interesting to assess the biological significance of cyano modification on rpsd.
while the current lack of available experimental data with regard to ptcms for salmonella typhimurium  prohibits us from validating predicted modifications presented herein, future salmonella typhimurium studies may confirm several of these putative modifications, leading to a greater understanding of the biology of this pathogenic organism.
annotation of post-translational proteolytic events
proteolytic cleavage plays an essential role in the control of numerous biological processes, including protein localization, fate and activity as well as the processing of cellular information.
however current high throughput genome annotation pipelines are blind to this information, any amount of which would clearly improve the quality of the genome annotation.
the low throughput and labor-intensive nature of edman degradation and two dimensional gel electrophoresis approaches make them incompatible with state of the art high throughput annotation pipelines.
using the high precision ms/ms data generated above, we highlight the use of proteomics data to identify a subset of post-translational proteolytic events in a high throughput label-free manner and include this additional layer of information to improve the quality of the genome annotation.
data were searched against the predicted genes in the stm  <dig> genome sequence using x!tandem  <cit>  and included  <dig> unique fully tryptic peptides ,  <dig> with one tryptic end , and  <dig> with no tryptic ends , identified at < 1% fdr.
in light of the high specificity of trypsin  <cit> , it is likely that peptides with either one or no tryptic termini are representative of proteolytic events.
we note that these peptides may also be generated by degradation of fully tryptic peptides due to hydrolysis during sample processing or to in-source decay during instrumental analysis, both of which introduce a potential source of ambiguity.
to address this concern, we employed a two step filtering procedure.
first, we only considered non- or partially-tryptic peptides that were not contained within a longer observed tryptic peptide, which reduced the non- or partially-tryptic peptide candidate list to  <dig> peptides.
second, we removed non- or partially-tryptic peptides contained within any other peptide, which further reduced the candidate peptide list for examining proteolytic events to  <dig> peptides .
while conservative, this two step filtering approach ensured we considered biologically generated non- and partially-tryptic peptides rather than experimental or analytical artifacts.
a potential drawback of this two-step approach is that identification of proteolytically processed proteins are typically made on the basis of single peptide identifications.
however this concern was mitigated by using high resolution and high mass measurement accuracy lc-ms/ms data.
if our hypothesis that non- or partially-tryptic peptides likely represent the results of possible proteolytic events were false, we would expect to see a relatively uniform distribution of non- or partially-tryptic peptides across the protein sequence.
to the contrary, when the frequency of the occurrence of peptides and the residue start positions between  <dig> and  <dig>  are plotted, two pronounced peaks are apparent, one representing residue start positions 1- <dig> and the other, representing residue start positions 21- <dig>
in figure  <dig>  the peak at residue start positions 1- <dig>  which is comprised almost exclusively  of peptides with residue start position  <dig>  is indicative of n-terminal methionine cleavage, a well-known post-translational modification recognized to be the major source of n-terminal amino acid diversity and thought to play an important role in controlling protein half-life  <cit> .
examination of the peptides with residue start position  <dig> revealed amino acids with small side chains  in the penultimate  position, with ser  and ala  most frequently observed , which agrees with established n-terminal methionine cleavage rules; require met as the p <dig> residue and amino acids with small side chains as the p1' residue   <cit> .
using this set of peptides we confirm cleavage of n-terminal methionine in  <dig> proteins .
the peak at residue start positions 21- <dig> is indicative of signal peptide cleavage, another well-known post-translational modification.
most signal peptides in gram-negative bacteria range between  <dig> and  <dig> amino acids in length, with an estimated average length of  <dig> amino acids, which is in agreement with our data  <cit> .
examination of the sequence immediately upstream of putative signal peptides  revealed a clear sequence motif  <cit>   that closely matches motifs used by signalp  <cit> , shown to be the most accurate in a recent comprehensive assessment of n-terminal signal peptides prediction methods  <cit> .
this provides additional support for using this characteristic set of peptides for identifying signal peptide cleavages.
signal peptides are essential for proper cellular function in both eukaryotes and prokaryotes, targeting proteins for secretion or for transportation to appropriate cellular locations.
for example in pathogenic bacteria, secretion of effector molecules is a central hallmark of the host-pathogen interaction in establishing a successful infection.
although algorithms such as signalp enable genome-based predictions of signal peptides, they almost always lack experimental verification.
this lack of verification is of particular concern in the highly sensitive area of recombinant protein production for human therapeutic use where inclusion of amino acids that are actually cleaved in the mature protein might elicit an immune response  <cit> .
using the current set of peptides we provide genome-scale experimental evidence for signal peptide cleavage for  <dig> proteins .
analysis of homologs in stm lt <dig> with > 99% identity by signalp revealed  <dig> of these proteins were predicted to possess a signal peptide .
collectively, these results demonstrate the utility of high accuracy ms/ms data for providing label-free high throughput genome-scale experimental confirmation of signal peptide cleavage identifications to improve the quality of the genome annotation.
we note that a recent study used a related approach to predict and validate signal peptides in the extracellular proteome of a microbial community  <cit> .
CONCLUSIONS
in this study, we reported the genomic sequence of the bacterial pathogen salmonella typhimurium   <dig> and demonstrated the use of ms-based proteomics to guide accurate primary genome annotation.
our proteomics data provided experimental confirmation for > 40% of the predicted protein-coding genes, and further improved the genome annotation with  <dig> start site corrections and the identification of  <dig> novel genes missed by gene finding programs, importantly these include some that appear to play a role in salmonella pathogenesis.
comprehensive analysis of post-translational processing events in stm  <dig> identified more than  <dig> signal peptide and n-terminal methionine cleavage events critical for protein function and also revealed a large and complex repertoire of post-translational chemical modifications, including those known to influences various cell processes and several novel chemical modifications.
a major impact of this work on genome annotation efforts is in demonstrating the utility of proteogenomics for high-throughput protein-level experimental annotation, validating and augmenting the in silico primary annotation.
even for a well annotated organism like salmonella with a high degree of homology to e. coli, we still uncovered a number of annotation errors as well as novel genes.
an additional impact of this work is further highlighted in the unique capability of proteogenomics to experimentally annotate in vivo post-translational processing events, increasingly recognized to play important roles in prokaryotic biology.
as more distantly related organism are sequenced in efforts to sample more branches of the tree of life, the level of accuracy for current gene prediction programs trained on proteobacteria datasets is expected to markedly decrease, and depending on the gc content of a genome annotation methods suffer accordingly.
thus, the ability of proteogenomics to provide direct protein-level evidence for a significant fraction of predicted protein coding genes will be of significant benefit as a complementary tool in any genome annotation effort.
in addition, the data used for proteogenomic annotations may already be generated as part of existing proteomics experiments and then only requires a different analysis method to incorporate the results.

 many aspects of modern biological research are dependent on accurate identification of the protein-coding genes in each genome, as well as the nature of the mature functional protein products, a process commonly referred to as genome annotation. with the exponential increase in the number of sequenced prokaryotic genomes afforded by advances in genome sequencing technologies over the last decade, present day prokaryotic genome annotation is essentially an automated high-throughput process that relies heavily on de novo gene prediction programs. while de novo gene prediction programs have significantly improved for prokaryotic genomes considerable challenges remain, such as determining the precise start and stop site of a gene, accurately predicting short genes, and determining a stop codon that represents an alternative amino acid rather than a true stop site. as efforts to sequence more branches of the tree of life expand, the level of accuracy for current gene prediction programs trained on proteobacteria datasets will markedly decrease, leading to an increase in incorrect predictions of protein-coding genes. compounding the issue is the lack of experimental evidence in support of predicted protein-coding regions for the overwhelming majority of annotated genomes. where available, experimental evidence is typically based on expressed rna sequences, such as from microarray or rna seq experiments. however, these genome-centric analyses do not independently and unequivocally determine whether a predicted protein-coding gene is translated into a protein or importantly provide any reliable information on post-translational processing. bottom-up proteomics offers the ability to directly measure peptides arising from expressed proteins representing the current best option for independently and unambiguously identifying at least an important subset of the protein-coding genes in a genome and can be used to experimentally validate gene annotations. in a bottom up approach, proteins within a complex mixture are typically digested with a protease, after which the resulting peptides are separated by chromatographic methods and then analyzed using tandem mass spectrometry. each ms/ms spectrum is a measure of fragment masses, ideally from a single peptide sequence of ~ 6- amino acids. this set of mass values is analogous to a'fingerprint' that identifies the peptide. interpretation of ms/ms peptide spectra is accomplished 1) by using algorithms such as x!tandem, sequest, or mascot to compare measured masses against a set of theoretical masses of possible protein sequences or 2) less commonly, by de novo analysis, which does not depend on any prior knowledge of the possible sequences. similar to searching ms/ms spectra against a set of predicted protein sequences, it is also possible to identify the protein-coding genes in a genome by searching ms/ms spectra against a six-frame translation of the genomic dna sequence, thereby precluding the inherent biases derived from gene prediction methods. we note that scaling up to an exponentially larger database, as a result of six-frame translation of a genomic dna sequence, makes searches slower by potentially orders of magnitude. additionally, it also results in increased possibility for false-positive identifications as the false discovery rate scales with the increasing database size, greatly decreasing sensitivity; twin challenges that can only be feasibly met with dedicated/sophisticated computing resources precluding routine use at present. bottom-up proteomics also provides an avenue for obtaining biologically relevant information about post-translational modifications. even for relatively simple biological systems such as prokaryotes, post-translational modification events are increasingly being recognized as important, but are poorly characterized with regard to sites or function. while ptm information gained from genome-scale ms/ms datasets stands to benefit biological understanding of bacterial organisms, significant technological challenges hamper the routine inclusion of this valuable information in primary annotations. this situation is exemplified by only a single report of genome-scale ms/ms datasets being used to comprehensively annotate post-translational modifications events in a genome sequence. however, the low resolution of the ms/ms datasets makes assignment of modifications, including crucially the site of modification, less confident and ill suited for routine use. for example, the dalton mass difference between gln and lys cannot be resolved in low resolution ms/ms spectra, which increases the number of possible peptide candidates to be matched and reduces confidence in assignments. on the other hand, the sequencing precision afforded by high resolution ms/ms spectra can resolve residues with small mass differences like gln and lys, which reduces the search space and thus the computational burden, and increases confidence in assignments. in this study, we employed a bottom-up proteomics approach supplemented with a recently described de novo sequencing methodology using high resolution and high mass measurement accuracy ms/ms data to accurately uncover the translational landscape and post-translational features of the bacterial pathogen salmonella enterica serovar typhimurium salmonella typhimurium is a leading cause of bacterial gastroenteritis and is widely used as a model to investigate basic genetic mechanisms as well as the interaction between bacterial pathogens and mammalian hosts. in spite of the clinical and basic science relevance of salmonella typhimurium there has been no comprehensive analysis undertaken to provide experimental support of its in silico-based genome annotation to facilitate systems-level analysis. our data provides protein-level experimental validation for approximately half of the predicted protein-coding genes in stm and suggests revisions to genes assigned incorrect translational start sites, including a potential novel alternate start codon. additionally, we uncovered non-annotated genes missed by gene prediction programs, as well as evidence suggesting a role for one of these novel orfs in salmonella pathogenesis. we also characterized post-translational features in the stm genome, including chemical modifications and proteolytic cleavages. we find that bacteria have a much larger and complex repertoire of chemical modifications than previously thought including several novel modifications. our in vivo proteolysis data identified more than signal peptide and n-terminal methionine cleavage events critical for protein function. contrary to the overwhelming majority of proteogenomics analyses that utilize proteomics data to improve the quality and completeness of previously annotated genomes, this study represents one of the first to utilize proteomics data "as part of a largely automated, high-throughput annotation process directly at the primary stage of genome annotation".  the genome of salmonella typhimurium 14028s was sequenced as described in the methods section and is composed of two replicons: a main chromosome and a plasmid with over 99% sequence homology to the salmonella typhimurium lt virulence plasmid pslt. the automated annotation pipeline at the j. craig venter institute was employed to identify genome features in the raw dna sequence, gather evidence for function of the features, and assign functional annotation. the main genome characteristics of stm are presented in table and the full list of predicted protein-coding genes is provided in additional file table s for the proteogenomic analysis described in this study we focused on chromosome-encoded genome features. low resolution liquid chromatography -ms/ms datasets were employed to experimentally validate gene annotations and high resolution lc-ms/ms datasets were employed to accurately annotate post-translational features as described below. to confirm and correct stm annotations and in particular to identify proteins specified by non-annotated genes, we searched low resolution ms/ms spectra from individual lc-ms/ms analyses against a six-frame translation of the stm genome sequence. these data comprise stm samples from different cell culture conditions designed to mimic various aspects of the non-infectious and infectious environments experienced by salmonella typhimurium. briefly, these included luria-bertani logarithmic and lb stationary phases, and two acidic minimal medium conditions, amm and amm. the mass spectrometry datasets employed in this proteogenomic annotation study have been previously described, detailing the annotated salmonella typhimurium proteome response to the above growth conditions. peptides with either one or two tryptic ends were filtered according to the parameters described in the methods section. as an additional confidence metric we only retained those filter-passing peptides that mapped to unique locations in the six-frame translation. peptides mapping to multiple locations in the six-frame translation introduce a potential source of ambiguity making it difficult to confidently validate individual orfs or correct annotation errors. a total of unique peptides that passed the above criteria were identified with a false discovery rate of < 1%. of these, peptides were assigned as being generated by two tryptic ends and with one tryptic end. experimental validation of predicted genes at the protein level for the majority of genes annotated as part of sequencing efforts there is no direct experimental evidence that the gene is translated into a protein. in cases where experimental evidence is available it is typically based on expressed rna sequences, such as from microarray or rnaseq experiments. it is clear however that expressed rna sequences cannot independently and unequivocally determine whether a predicted protein-coding gene is translated into a protein. thus knowing that a gene or part of it in the relevant frame is being made is useful information. multiple unique peptides mapping to a single gene provide compelling evidence for the expression of the product encoded by that particular gene. as such, we matched the identified unique peptides to the predicted chromosomal genes in the stm genome sequence to validate automated gene predictions at the protein level. the predicted genes covered most of the observed peptides. conservatively, a gene product was confirmed expressed at the protein level only when a minimum of two unique peptides mapped to the particular gene. using this criterion, our proteomics data validated of the annotated chromosomal orfs in the stm genome sequence at the protein level, providing experimental evidence for the products of these genes. figure shows an example of a predicted gene whose length is covered by multiple identified peptides providing experimental evidence for its expression at the protein level. given the limited number of possible tryptic peptides available for protein identification in small orfs, the above criteria would presumably preclude the confident identification of novel small orfs. the ~44% level of proteome coverage is in line with, and in most published cases exceeds, results obtained in both typical proteomics experiments and previous proteogenomic analyses. typical coverage is in the 30% range for the annotated orfs. however, multiple factors potentially prevent 100% coverage, including extensive post-transcriptional regulation that makes transcripts rather poor guides for protein expression. here we have used four differing growth conditions including typical laboratory conditions and an infection-like environment. however, not all possible growth conditions for salmonella typhimurium can be efficiently sampled and it is likely that certain orfs require specific conditions for expression, such as intracellular growth in host cells. in addition, various technical aspects associated with our experiments may have precluded detection, such as incompatibility with buffers for soluble protein extraction. the two peptide criterion may also negatively impact coverage of small proteins present in low concentrations. however it is possible that application of additional protein and peptide separation methods could enable improved coverage of low abundance proteins. refinement of predicted gene structures: start sites determining the correct start position of a gene remains a challenge for current gene prediction algorithms. in a recent re-analysis of annotated prokaryotic genomes, nielsen and krogh observed that in some genomes up to 60% of the genes may have been annotated with a wrong start codon, especially in gc-rich genomes. accurate start site predictions better define intergenic spaces that may encode promoters and regulatory binding sites, which are critical elements in studies of transcriptional regulation. cellular localization signals also are contained in start sites, which makes accurate start site predictions important for accurately determining the localization of proteins within a cell. peptides that map to genomic regions within bp upstream of previously annotated genes and in the same translational reading frame represent evidence supporting extensions of their predicted start sites. we note that this approach has been described in detail in a number of recent publications. additionally peptides that span the start site of previously annotated genes also represent evidence supporting extensions of their predicted start sites. to this end, we examined the identified peptides for experimental evidence supporting extension of start sites in each of the predicted genes, and where possible proposed a new start codon. peptides spanned the start site of previously annotated genes, and peptides mapped to genomic regions within 200bp upstream of previously annotated genes. overall, of the peptides mapped to genomic regions that served as experimental evidence supporting extension of start sites. we restricted our correction of start sites to predicted genes confirmed at the protein level, which yielded a final candidate list of peptides that correspond to genes requiring n-terminal extension/start site correction. new start sites were largely defined by the first methionine amino acid and/or start codon encountered upstream of peptides mapped to the genomic region within bp upstream of their previously annotated start sites. figures and highlight examples depicting the utility of proteomics data for start site correction. orf is a member of the universal stress protein family of proteins with biological roles in motility, adhesion and coping with stress. in our proteomics data we observed several peptides upstream of orf in the same reading frame and within bp of the predicted start site, i.e., evidence supporting extension of the predicted start site. furthermore, three of these peptides span the previously predicted start site, providing additional evidence in support of refining the start site. orf is the nadh-dependent enoyl reductase from the type ii bacterial fatty acid biosynthesis pathway, as inferred from the primary in silico genome annotation, a validated but currently underexploited target for drug discovery. similarly, we observed several peptides that overlap this orf including one peptide that spanned the currently predicted gene start. the accurate experimental delineation of the structure of this clinically relevant gene represents an important step in facilitating ongoing drug development efforts. while beyond the scope of this study, we remark that protein expression and purification coupled with edman degradation represents a useful orthogonal avenue to validate at the protein level the corrected assignments proposed. in prokaryotes the initiation of translation is typically mediated by the start codon atg. in addition gtg and ttg are also used as alternative start codon. studies in e. coli have estimated the frequency of start codon usage as atg 83%, gtg 14% and ttg 3%. however a very small number of studies have also reported ctg, att, ata, and atc codons to function as rare non-canonical translation initiators. interestingly our proteomics data showed multiple peptides that overlap orf including one peptide that spanned the currently predicted gene start, providing evidence in support of extending the start site. the peptide, giepmaltkaemseylfdklglskr, is immediately adjacent to a stop codon with another stop codon a further seven amino acids upstream making the possibility that the peptide results from a stop codon read through unlikely. considering this, the first amino acid in the peptide sequence glycine would presumably be the translation initiator. glycine is coded for by the triplet codon ggg. however, it is well known that even if an alternative start codon is used as the translation initiator, it gets translated as methionine. thus we examined the possibility of a frame-shift involving the preceding orf phet, coded in a different frame, as a potential mechanism to explain this observation. however, the orf phet terminates with the "...aslrd-stop" amino acid sequence conserved across several related salmonella and e. coli strains. it is clear that further studies beyond the scope of the present work will be required to resolve and or confirm this interesting observation. the peptide giepmaltkaemseylfdklglskr is defined as fully tryptic, having a native n-terminus and a c-terminal arginine, and was observed in three independent lc-ms/ms analyses with minimum sequest® peptide identification scores across the three independent lc-ms/ms analyses of xcorr ≥, Δcn ≥, and peptideprophet™ probability ≥. a representative ms/ms spectrum of the peptide giepmaltkaemseylfdklglskr is shown in figure while we do not observe any fragments ions corresponding to proline directed fragmentation, the near complete y ion series and extensive sequence coverage when considering both b and y ion series provides additional confidence to the annotation. identification of novel genes the identification of protein-coding genes in eukaryotic genomes is complicated by the high frequency of alternative splicing in most eukaryotic genes. additionally the small fraction of protein-coding genes that comprise eukaryotic genomes make the identification of coding sequences against the ubiquitous background of non-coding sequences difficult. in contrast, the usual absence of introns and the compact nature of prokaryotic genomes make the identification of all possible orfs longer than a chosen threshold in a dna sequence a relatively straightforward computational exercise. this view, in particular of bacterial genomes, has lead to the assumption that computationally derived coding sequences completely describe the entire coding capacity of a bacterial genome. however, structural annotation of prokaryotic genome sequences by predicting coding sequences is far from being a trivial matter. a number of recent studies used proteomics data to identify novel protein-coding genes in prokaryotic genomes that had been missed by de-novo gene finding programs. peptides that map to genomic regions outside the boundaries of predicted genes are evidence suggestive of the presence of novel genes missed by gene finding programs. by matching the identified peptides to the predicted chromosomal genes in the stm genome sequence, we detected peptides mapped to regions falling outside the boundaries of known protein coding genes. we further refined the list of peptides by excluding the peptides that mapped to genomic regions within bp upstream of predicted genes, i.e., peptides that are indicative of n-terminal extensions, which yielded a final candidate list of peptides. these peptides represent experimental evidence for the presence of novel genes missed by gene finding programs. using the set of intergenic peptides, we defined novel genes that had been missed by gene prediction methods. note that novel genes were confirmed as detected and expressed at the protein level only when a minimum of two peptides mapped to that particular novel gene. of particular interest was the novel orf c1368_1:795109- defined by five peptides that map to the genomic region to where no gene had been predicted. protein blast analysis of the genomic region where the five peptides mapped to against the non-redundant protein sequences database revealed a single significant hit to a phage protein in salmonella typhimurium strain d. interestingly, examination of our experimental ms/ms data showed that this novel orf c1368_1:795109- was highly expressed under growth in acidic minimal media relative to growth under standard laboratory conditions. growth in amm has been shown to approximate the environment found within the salmonella-containing vacuoles observed in infected host macrophages, and we among others have shown that this media induces the expression of the salmonella pathogenicity island -encoded type-iii secretion system, a system critical for virulence and intramacrophage survival. thus proteins co-regulated with known virulence proteins under such environmental conditions are likely to play a role in salmonella typhimurium pathogenesis. indeed phage proteins are known to play an important role in salmonella typhimurium pathogenesis further supporting our observation. the full repertoire of factors employed by salmonella typhimurium to establish a successful infection is still unknown, presumably because they remain undetected due to a number of reasons including being missed by annotators; thus the enumeration of all proteins in the salmonella typhimurium genome, especially those co-regulated with known virulence factors, as presented above is an important step in understanding the mechanisms of salmonella typhimurium pathogenesis. assessing the performance of proteogenomic annotation manual curation has become a luxury reserved for a few organisms and the majority of newly sequenced genomes only receive a single round of computational annotation with no additional manual refinement, therefore the utility of proteogenomics lies in its application at the primary stage of genome annotation to improve the quality and completeness of the automated genome annotation. a version of the stm genome annotated using a combination of computational methods and human annotators, i.e. refined annotation, released while this manuscript was in preparation allowed us to assess the performance of proteogenomic annotation. a comparison of the new start sites for the genes proposed by the proteomics data to the start sites for the same genes in the refined stm genome annotation revealed an overlap of genes where start sites matched exactly. of the remaining genes, had proteomics-suggested start sites that were downstream of those determined by refined annotation. while the proteomics data suggest a shorter orf than indicated in the refined annotation, homology analysis supports the refined annotation. note that deeper proteome coverage may improve matches to the refined annotation. the remaining three genes had proteomics-suggested start sites that were upstream of start sites in the refined annotation, which is tangible experimental evidence that suggests a longer orf than indicated in the refined annotation, i.e., correcting the refined annotation. in summary of start site corrections proposed by our proteogenomic analysis in the primary annotation reported in this study were in line with the refined annotation. the remaining three start site corrections suggested represent corrections to both the automated and refined annotation. our proteogenomic analysis also identified novel genes missed by gene prediction methods. a comparison of the novel genes proposed by proteomics data to the refined stm genome annotation revealed of the novel genes were also identified and annotated similarly in the refined annotation. this result highlights the power of proteomics data to identify relevant novel protein-coding genes. the three remaining proteomics-identified genes missed by de novo gene finding programs and human annotators represent a correction and improvement to both the current automated and refined annotations. annotation of complex post-translational chemical modifications while a number of groups have used genome-scale ms/ms data to confirm predicted bacterial genes at the protein level, as well as identify new genes and correct gene prediction, there is only a single report of genome-scale ms/ms data being used for comprehensive "unrestricted" analysis and annotation of post-translational chemical modifications in a bacterial system. we note however a number of recent studies focused on characterizing ribosomal protein modifications. as relatively little is known about ptcms in bacteria, even for intensively studied model organisms such as e. coli and salmonella, any ptcm information gained from genome-scale ms/ms data would aid biological understanding of bacterial organisms. we have recently described a de novo sequencing approach, using high resolution and high mass measurement accuracy ms/ms data, for the accurate discovery of unknown or unexpected ptcms of proteins. here we apply the de novo-ustags approach to analyze high resolution lc-ms/ms datasets for ptcms in the stm genome. these datasets represented samples that had been grown in a variety of cell culture conditions. using the approach illustrated above, we determined mass shifts for ustags in ms/ms spectra that represented proteins. we estimate the false discovery rate of ptcm analysis to be < 1%. utilizing in-house developed software and a list of unimod modifications, including snp substitutions, http://www.unimod.org we inferred hypothetical explanations for observed mass shifts in ~92% of the spectra in which modifications were detected, several of which had multiple potential chemical explanations. where applicable observation of "differential" fragments were used to resolve ambiguity of assignments between snps and other modifications of similar mass defect as described below and in the methods and additional file the remaining spectra in which modifications were detected but where no plausible modification combination of up to two of the selected unimod modifications could explain observed mass shift for prefix or suffix sequence represent novel previously un-described ptcms and are listed in additional file table s5e. to obtain lower limit estimates on the number of distinct ptcms, we binned different mass shifts and inferred a total of distinct modifications mass-shifts, each with multiple potential chemical explanations according to unimod. this represents a much larger and complex repertoire of chemical modifications than previously thought existed in bacteria. note that this only considered mass-shifts for which a potential chemical explanation was found in unimod, thus most likely an underestimate. nevertheless this estimate represents a much larger number of modification types than can be considered by commonly used, but often restrictive ptcm search algorithms such as sequest, x!tandem, and mascot. the modification containing ms/ms spectra with hypothetical explanations for the observed mass shifts were ranked using a peptide identification scoring function and based on the identification scores we obtained unambiguous explanations, with regards to type, number and site of modification, for observed mass shifts in ms/ms spectra. we also established confident assignment of number and type of modification to explain observed mass shifts in an additional ms/ms spectra; however site of modification could not be unambiguously derived from spectral evidence. among the modification types observed were those known to result from sample preparation, including carbamylation and carbamidomethyl, as well as those that can occur both in vitro and in vivo, such as methionine oxidation and asparagine deamidation. as no methods are currently available for distinguishing between in vitro and in vivo modifications, to identify ptcms in stm that are biologically relevant we assumed such modifications would be conserved across closely related organisms. using this approach we report a number of ptcms of biological significance previously unappreciated in stm. methylation of ribosomal proteins has been suggested to modulate the intra- or intermolecular interactions of the methylated ribosomal proteins or affects their affinity for rna, and, thus, influences various cell processes, including ribosome assembly and translation accuracy. single methylation of the e. coli ribosomal protein l7/ has been reported and localized to k, while the ribosomal protein l has been reported as being methylated at q in e. coli. in the present study we observe methylation of the e. coli ribosomal protein l7/ homolog in stm orf at the same location, k and methylation of the ribosomal protein l ortholog in stm orf at the exact same position, q suggesting a similar regulatory and/or structural role in stm as in e. coli. methylation of the translation elongation factor tu at position k has been reported, and suggested as a mechanism for'fine tuning' of tufb-trna inter-molecular interactions. we observed the same modification at k of the stm protein orf the homologous position to k of the e. coli protein, suggesting a similar functional role in stm as in e. coli. besides those ptcms with functional inferences from comparative analysis we observed additional salmonella typhimurium ptcms of interest. for example, figure shows the detection of a cyano modification at residue cys of the salmonella typhimurium protein ribosomal protein s. note that the cyano modification is classified in unimod as a post-translational modification and not a chemical derivative/artifact. this to our knowledge is the first report of an in vivo cyano modification in salmonella. previous studies in e. coli have reported on the effects of cyanate on the different catalytic activities of carbamyl phosphate synthetase. it will be interesting to assess the biological significance of cyano modification on rpsd. while the current lack of available experimental data with regard to ptcms for salmonella typhimurium prohibits us from validating predicted modifications presented herein, future salmonella typhimurium studies may confirm several of these putative modifications, leading to a greater understanding of the biology of this pathogenic organism. annotation of post-translational proteolytic events proteolytic cleavage plays an essential role in the control of numerous biological processes, including protein localization, fate and activity as well as the processing of cellular information. however current high throughput genome annotation pipelines are blind to this information, any amount of which would clearly improve the quality of the genome annotation. the low throughput and labor-intensive nature of edman degradation and two dimensional gel electrophoresis approaches make them incompatible with state of the art high throughput annotation pipelines. using the high precision ms/ms data generated above, we highlight the use of proteomics data to identify a subset of post-translational proteolytic events in a high throughput label-free manner and include this additional layer of information to improve the quality of the genome annotation. data were searched against the predicted genes in the stm genome sequence using x!tandem and included unique fully tryptic peptides, with one tryptic end, and with no tryptic ends, identified at < 1% fdr. in light of the high specificity of trypsin, it is likely that peptides with either one or no tryptic termini are representative of proteolytic events. we note that these peptides may also be generated by degradation of fully tryptic peptides due to hydrolysis during sample processing or to in-source decay during instrumental analysis, both of which introduce a potential source of ambiguity. to address this concern, we employed a two step filtering procedure. first, we only considered non- or partially-tryptic peptides that were not contained within a longer observed tryptic peptide, which reduced the non- or partially-tryptic peptide candidate list to peptides. second, we removed non- or partially-tryptic peptides contained within any other peptide, which further reduced the candidate peptide list for examining proteolytic events to peptides. while conservative, this two step filtering approach ensured we considered biologically generated non- and partially-tryptic peptides rather than experimental or analytical artifacts. a potential drawback of this two-step approach is that identification of proteolytically processed proteins are typically made on the basis of single peptide identifications. however this concern was mitigated by using high resolution and high mass measurement accuracy lc-ms/ms data. if our hypothesis that non- or partially-tryptic peptides likely represent the results of possible proteolytic events were false, we would expect to see a relatively uniform distribution of non- or partially-tryptic peptides across the protein sequence. to the contrary, when the frequency of the occurrence of peptides and the residue start positions between and are plotted, two pronounced peaks are apparent, one representing residue start positions 1- and the other, representing residue start positions 21- in figure the peak at residue start positions 1- which is comprised almost exclusively of peptides with residue start position is indicative of n-terminal methionine cleavage, a well-known post-translational modification recognized to be the major source of n-terminal amino acid diversity and thought to play an important role in controlling protein half-life. examination of the peptides with residue start position revealed amino acids with small side chains in the penultimate position, with ser and ala most frequently observed, which agrees with established n-terminal methionine cleavage rules; require met as the p residue and amino acids with small side chains as the p1' residue. using this set of peptides we confirm cleavage of n-terminal methionine in proteins. the peak at residue start positions 21- is indicative of signal peptide cleavage, another well-known post-translational modification. most signal peptides in gram-negative bacteria range between and amino acids in length, with an estimated average length of amino acids, which is in agreement with our data. examination of the sequence immediately upstream of putative signal peptides revealed a clear sequence motif that closely matches motifs used by signalp, shown to be the most accurate in a recent comprehensive assessment of n-terminal signal peptides prediction methods. this provides additional support for using this characteristic set of peptides for identifying signal peptide cleavages. signal peptides are essential for proper cellular function in both eukaryotes and prokaryotes, targeting proteins for secretion or for transportation to appropriate cellular locations. for example in pathogenic bacteria, secretion of effector molecules is a central hallmark of the host-pathogen interaction in establishing a successful infection. although algorithms such as signalp enable genome-based predictions of signal peptides, they almost always lack experimental verification. this lack of verification is of particular concern in the highly sensitive area of recombinant protein production for human therapeutic use where inclusion of amino acids that are actually cleaved in the mature protein might elicit an immune response. using the current set of peptides we provide genome-scale experimental evidence for signal peptide cleavage for proteins. analysis of homologs in stm lt with > 99% identity by signalp revealed of these proteins were predicted to possess a signal peptide. collectively, these results demonstrate the utility of high accuracy ms/ms data for providing label-free high throughput genome-scale experimental confirmation of signal peptide cleavage identifications to improve the quality of the genome annotation. we note that a recent study used a related approach to predict and validate signal peptides in the extracellular proteome of a microbial community.  in this study, we reported the genomic sequence of the bacterial pathogen salmonella typhimurium and demonstrated the use of ms-based proteomics to guide accurate primary genome annotation. our proteomics data provided experimental confirmation for > 40% of the predicted protein-coding genes, and further improved the genome annotation with start site corrections and the identification of novel genes missed by gene finding programs, importantly these include some that appear to play a role in salmonella pathogenesis. comprehensive analysis of post-translational processing events in stm identified more than signal peptide and n-terminal methionine cleavage events critical for protein function and also revealed a large and complex repertoire of post-translational chemical modifications, including those known to influences various cell processes and several novel chemical modifications. a major impact of this work on genome annotation efforts is in demonstrating the utility of proteogenomics for high-throughput protein-level experimental annotation, validating and augmenting the in silico primary annotation. even for a well annotated organism like salmonella with a high degree of homology to e. coli, we still uncovered a number of annotation errors as well as novel genes. an additional impact of this work is further highlighted in the unique capability of proteogenomics to experimentally annotate in vivo post-translational processing events, increasingly recognized to play important roles in prokaryotic biology. as more distantly related organism are sequenced in efforts to sample more branches of the tree of life, the level of accuracy for current gene prediction programs trained on proteobacteria datasets is expected to markedly decrease, and depending on the gc content of a genome annotation methods suffer accordingly. thus, the ability of proteogenomics to provide direct protein-level evidence for a significant fraction of predicted protein coding genes will be of significant benefit as a complementary tool in any genome annotation effort. in addition, the data used for proteogenomic annotations may already be generated as part of existing proteomics experiments and then only requires a different analysis method to incorporate the results. 
our in vivo proteolysis data identified more than signal peptide and n-terminal methionine cleavage events critical for protein function. we also characterized post-translational features in the salmonella genome, including chemical modifications and proteolytic cleavages. we find that bacteria have a much larger and more complex repertoire of chemical modifications than previously thought including several novel modifications. furthermore, gene prediction programs provide no information on biologically important post-translational processing events critical for protein function. additionally, we uncovered non-annotated genes missed by gene prediction programs, as well as evidence suggesting a role for one of these novel orfs in salmonella pathogenesis. the data provide protein-level experimental validation for approximately half of the predicted protein-coding genes in salmonella and suggest revisions to several genes that appear to have incorrectly assigned translational start sites, including a potential novel alternate start codon. this work highlights several ways in which application of proteomics data can improve the quality of genome annotations to facilitate novel biological insights and provides a comprehensive proteome map of salmonella as a resource for systems analysis. we experimentally annotated the bacterial pathogen salmonella typhimurium using "shotgun" proteomics to accurately uncover the translational landscape and post-translational features.
 complete and accurate genome annotation is crucial for comprehensive and systematic studies of biological systems. however, determining protein-coding genes for most new genomes is almost completely performed by inference using computational predictions with significant documented error rates. furthermore, gene prediction programs provide no information on biologically important post-translational processing events critical for protein function.  we experimentally annotated the bacterial pathogen salmonella typhimurium using "shotgun" proteomics to accurately uncover the translational landscape and post-translational features. the data provide protein-level experimental validation for approximately half of the predicted protein-coding genes in salmonella and suggest revisions to several genes that appear to have incorrectly assigned translational start sites, including a potential novel alternate start codon. additionally, we uncovered non-annotated genes missed by gene prediction programs, as well as evidence suggesting a role for one of these novel orfs in salmonella pathogenesis. we also characterized post-translational features in the salmonella genome, including chemical modifications and proteolytic cleavages. we find that bacteria have a much larger and more complex repertoire of chemical modifications than previously thought including several novel modifications. our in vivo proteolysis data identified more than signal peptide and n-terminal methionine cleavage events critical for protein function.  this work highlights several ways in which application of proteomics data can improve the quality of genome annotations to facilitate novel biological insights and provides a comprehensive proteome map of salmonella as a resource for systems analysis. gene annotationproteomicspost-translational modifications 