although decades have passed since most cellular organelles were initially characterized by microscopy and subcellular fractionation , a complete catalogue of the proteins in each organelle has yet to be obtained . whereas genomics provides a list of potential proteins encoded by an organism 's genome , data from proteomic analysis can provide the ' rosetta stone ' that allows assignment of specific proteins to different subcellular structures . the most informative proteomic analysis requires highly purified organelle ( sub)fractions , yet subcellular fractionation is notorious for cross - contamination . comparative analysis of organellar subfractions can potentially circumvent this problem by providing a rational basis for distinguishing bona fide organelle components from contaminants . here , we scrutinize the use of such an approach to survey the integral membrane proteins of the nuclear envelope . the nuclear envelope is a double - membrane system , continuous with the endoplasmic reticulum ( er ) , that encloses the nuclear contents ( figure 1 ) . it is perforated by nuclear pore complexes ( npcs ) , large supramolecular assemblies that mediate nucleo - cytoplasmic traffic . in higher eukaryotes , the nuclear envelope is lined by a protein meshwork , the nuclear lamina , which is an attachment site for npcs   and chromatin . in contrast , the inner nuclear membrane ( inm ) contains a set of unique integral membrane proteins , many of which bind to lamins ( the predominant proteins of the nuclear lamina ) and/or to chromatin . a quarter of a century of biochemical , cell biological , and genetic approaches has identified several integral inm proteins in higher eukaryotes , including lamin - b receptor ( lbr ) , lamina - associated polypeptides ( laps ) 1 and 2 , man1 , nurim , and emerin . most of these proteins and several novel components have now been detected in a single proteomics study of nuclear envelopes . many methods have been developed to isolate nuclear and nuclear - envelope fractions from eukaryotic cells . these fractions are invariably contaminated by other cytoplasmic organelles and filaments , however . moreover , the close interactions between the npcs , nuclear lamina , inm and chromatin make it impossible to cleanly separate these components . proteomics , which couples mass - spectrometric analysis of proteins with mining of the protein databases derived from genomics , can potentially identify most proteins in a nuclear envelope fraction , but it can not distinguish nuclear envelope components from contaminants . dreger and colleagues   used comparative proteomics of different nuclear envelope subfractions isolated from cultured neuroblastoma cells to identify integral proteins of the inm . they took advantage of previous findings that the nuclear lamina is insoluble in both nonionic detergent and salt , and that many integral proteins of the inm remain associated with the lamina after detergent or salt extraction . they therefore separated proteins from three different nuclear envelope subfractions on two - dimensional gels , excised protein spots from the gels , cleaved the proteins within each spot with protease , and analyzed the resulting peptides by maldi - tof ( matrix - assisted laser - desorption / ionization time - of - flight ) mass spectrometry . in one extraction , nuclear envelopes were treated with the detergent triton x-100 to solubilize proteins of the outer nuclear membrane and er . the triton x-100 pellet ( ' detergent - resistant ' in table 1 ) , which contains the nuclear lamina , is enriched in lamin - binding inm proteins . a second pellet was obtained by salt extraction of nuclear envelopes ( ' salt - resistant ' ; table 1 ) and is expected to be similarly enriched in the lamina , but with different contaminants removed . a third extraction involved treatment of nuclear envelopes with a urea / carbonate solution and yields a pellet ( ' chaotrope - resistant ' ; table 1 ) containing integral membrane proteins of the inm , outer nuclear membrane and er ( plus contaminating organelles ) . on the basis of the logic discussed above , integral membrane proteins ( chaotrope pellet ) that were also found in the detergent and salt pellets were good candidates for novel inm proteins . this analysis   identified most previously characterized inm proteins , as well as four novel integral proteins . two of the novel four were splice variants of the lamina - associated protein lap2 , previously observed only as mrnas . the identification of unique peptides , including some that overlap predicted splice junctions between exons , confirmed the presence of three previously observed (  ,  ,  ) and two novel (  ,  ) lap2 isoforms . the remaining two lap2 isoforms that had been identified as mrnas (  ,  ' ) were not observed ( although all lap2 isoforms contain chromatin and/or lamin - binding domains that predict their targeting to the nuclear envelope ) . lap2 , a splice variant lacking a transmembrane domain , was absent from the chaotrope - resistant fraction , supporting the logic of the comparative approach . the third novel inm protein to be identified was a homolog of unc84a , a caenorhabditis elegans protein previously localized to the nuclear envelope . the fourth protein , luma , was completely novel and includes no known domains and so has no predicted functions . dreger and colleagues   did not detect lap1 , although it has been observed in a variety of mammalian cell types . this could result from its loss during fractionation , its absence from the neuroblastoma line examined , or other technical problems ( for example , only about 75% of the protein spots seen on the two - dimensional gel yielded assignments by mass spectrometry ) . low abundance is also a potential problem . whereas lamins are present at millions of copies per cell , previously characterized inm proteins are far less abundant . notwithstanding the high sensitivity of mass spectrometry , minor proteins may go undetected . just as local dialect modifications can limit some translations using the rosetta stone , atypical protein behaviors can limit comparative proteomic approaches . each comparative approach is tailored by knowledge of the fractionation behavior of previously characterized components of an organelle . thus , in comparing the proteins appearing in the three nuclear envelope fractions , dreger and colleagues   found that all well characterized intranuclear proteins were absent from the chaotrope - resistant fraction , and most known inm proteins were in all three fractions . emerin , which is known to bind lamins , was absent from the detergent - resistant fraction , and lbr , the sequence of which includes seven transmembrane segments , was detected only in the detergent - resistant fraction and not in the chaotrope- or salt - resistant fractions ( table 1 ) . three of the novel proteins identified by the study were in all three fractions , but luma was absent from the salt - resistant fraction . thus , luma would have been missed if a requirement that novel inm proteins appear in all three fractions had been enforced . atypical behavior of contaminants can also limit the effectiveness of comparative approaches . in the dreger study , some known proteins with clearly cytoplasmic localizations and functions also appeared in all three pellets , including mitochondrial proteins ( f1-atp - synthase  ) and cytoskeletal ones ( actin and tubulin ) . this underscores the need to complement the proteomic identification of new organellar components with other approaches , including direct localization . thus , to ensure that luma and the unc84a homolog were not contaminants , their cdnas were isolated , epitope - tagged , and transfected into cos7 cells , to confirm nuclear - envelope targeting . rout and colleagues , when analyzing the yeast npc by proteomics , used the additional criterion of enrichment with the npc to distinguish contaminants . candidate proteins from an npc - enriched fraction were epitope - tagged at their genomic loci . only when the majority of tagged protein was found to reside at the npc ( by immunogold electron microscopy , immunofluorescence microscopy , and co - fractionation with known npc proteins ) was it considered a true npc component . some proteins have multiple cellular localizations , however , and this criterion eliminated , for example , the sec13 protein , which is involved in protein translocation across the er membrane yet has a second function at the npc . even with the four novel inm proteins demonstrated by the dreger study , we predict that many inm proteins remain to be discovered ; some of these could be amongst the 25% of protein spots that eluded analysis . for example , unc84a appears in all c. elegans tissues , but unc83 , an inm protein that interacts with unc84a , appears in only a subset . nuclear envelope proteome analyses of different tissues may uncover a mammalian unc83 homolog and other tissue - specific inm proteins . indeed , a novel tissue - specific inm protein was just discovered . this protein , named myne-1 , binds lamins and appears specifically in myocytes . applying proteomics directly to complex protein mixtures , rather than restricting analysis to the proteins recovered from gels , should increase the number of proteins detected . moreover , this method could estimate the relative amounts of different proteins in a subcellular compartment by determining the relative peptide quantities after protease cleavage . post - translationally modified peptides could be identified by comparing peptide profiles with or without treatments that remove the modifications ( for example , chemical removal of sugars ) ; and a combination of this type of approach with relative quantitation could provide information on the proportion of a protein in an organelle that is modified . by analysis of complex mixtures , the protein profiles of organelles in different functional states or extracted with different ionic strengths can be compared more comprehensively and rapidly than with classical approaches . in the past two years , organellar proteomics has also profiled mitochondrial , chloroplast , and nucleolar   proteomes , uncovered minor golgi proteins , and compared functional states of the golgi . the speed of proteomics can produce petabytes of data far faster than we can analyze them . it thereby allows a wider range of comparative analyses than has so far been possible . the roads opened by comparative proteomics will one day provide a complete map of all the cellular proteins in each organelle in each tissue at each stage of development . through comparison of this map with datasets generated from people with particular diseases , proteomics the opening of the nuclear envelope by proteomics has already identified new inm proteins and produced a catalogue of yeast npc proteins . schematic of the nuclear envelope . the outer nuclear membrane ( onm ) and inner nuclear membrane ( inm )
proteomic studies have the potential to comprehensively define the composition of organelles but are limited by the organellar cross - contamination that arises during subcellular fractionation . 
 comparative proteomics of organellar subfractions can mitigate these problems , as demonstrated by a recent study involving the nuclear envelope .