Global Analysis of Gel Mobility of Proteins and Its Use in Target Identification

Abstract

SDS-PAGE is a basic method that has long been used for separation of proteins according to their molecular sizes. Despite its simplicity, it provides information on characteristics of proteins beyond their molecular masses because gel mobility of proteins often reflects their physicochemical properties and post-translational modifications. Here we report on a global analysis of gel mobility of the proteome, which we term the “mobilitome,” covering 93.4% of the fission yeast proteome. To our surprise, more than 40% of proteins did not migrate to their calculated positions. Statistical analyses revealed that the discrepancy was largely dependent on the hydrophobicity of proteins. This experimental data set, with a high coverage rate of real mobility, made it feasible to identify proteins detected on the gel without using any specialized techniques. This approach enabled us to detect previously unknown post-translational modifications of a protein; for example, we revealed that eIF5A is novel substrate of a Sir2-related deacetylase Hst2. Furthermore, we concomitantly identified twelve acetylated and eight methylated proteins using specific anti-acetylated and anti-methylated lysine antibodies, most of which had not been known to be subject to the modifications. Thus, we propose the general usefulness of the mobilitome and electrophoresis-based methodology for the identification and characterization of proteins detected on the gel. SDS-PAGE is a basic method that has long been used for separation of proteins according to their molecular sizes. Despite its simplicity, it provides information on characteristics of proteins beyond their molecular masses because gel mobility of proteins often reflects their physicochemical properties and post-translational modifications. Here we report on a global analysis of gel mobility of the proteome, which we term the “mobilitome,” covering 93.4% of the fission yeast proteome. To our surprise, more than 40% of proteins did not migrate to their calculated positions. Statistical analyses revealed that the discrepancy was largely dependent on the hydrophobicity of proteins. This experimental data set, with a high coverage rate of real mobility, made it feasible to identify proteins detected on the gel without using any specialized techniques. This approach enabled us to detect previously unknown post-translational modifications of a protein; for example, we revealed that eIF5A is novel substrate of a Sir2-related deacetylase Hst2. Furthermore, we concomitantly identified twelve acetylated and eight methylated proteins using specific anti-acetylated and anti-methylated lysine antibodies, most of which had not been known to be subject to the modifications. Thus, we propose the general usefulness of the mobilitome and electrophoresis-based methodology for the identification and characterization of proteins detected on the gel. Gel mobility analysis of proteins often provides useful information on their physicochemical properties and post-translational modifications and can sometimes provide important insights into the function and regulation of a given protein. SDS-PAGE has been long and widely used for separation and size determination of proteins. In the early 1960s, polyacrylamide gels became appreciated as a convenient and versatile alternative to starch gels (1Ornstein L. Ann. N. Y. Acad. Sci. 1964; 121: 321-349Crossref PubMed Scopus (3332) Google Scholar, 2Davis B.J. Ann. N. Y. Acad. Sci. 1964; 121: 404-427Crossref PubMed Scopus (15961) Google Scholar). It was subsequently demonstrated that in the presence of SDS, proteins migrate according to the molecular weight of their polypeptide chains (3Shapiro A.L. Vinuela E. Maizel Jr., J.V. Biochem. Biophys. Res. Commun. 1967; 28: 815-820Crossref PubMed Scopus (2653) Google Scholar, 4Weber K. Osborn M. J. Biol. Chem. 1969; 244: 4406-4412Abstract Full Text PDF PubMed Google Scholar). However, the band resolution of the technique was relatively low. Therefore, this system, which uses only one phosphate buffer for the tanks and the gel, has lost its popularity despite its simplicity. Meanwhile, Laemmli modified the Ornstein and Davis system by adding SDS to the Tris/glycine buffer, in which a nonrestrictive large pore gel called a stacking gel is put on the top of a separating gel, and showed that proteins could be reliably separated with high resolution (1Ornstein L. Ann. N. Y. Acad. Sci. 1964; 121: 321-349Crossref PubMed Scopus (3332) Google Scholar, 2Davis B.J. Ann. N. Y. Acad. Sci. 1964; 121: 404-427Crossref PubMed Scopus (15961) Google Scholar, 5Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). As is well known, the Laemmli modification of the Ornstein and Davis system has become a mainstream technique for protein separation. Identification of a protein detected on an SDS-polyacrylamide gel is a critical step for biochemical studies. Recent advances in analytical techniques, such as mass spectrometry, as well as the accumulation of genome information, have enabled systematic identification of proteins on a gel. Despite the high sensitivity of mass spectrometry, however, it is still difficult to identify the proteins when they are present in low abundance, highly modified, or impure. Therefore, in addition to these forward proteomic techniques, a reverse proteomic approach based on the set of all identified genes would be useful. The genome sequence of the fission yeast Schizosaccharomyces pombe was completed in 2002 (6Wood V. Gwilliam R. Rajandream M.A. Lyne M. Lyne R. Stewart A. Sgouros J. Peat N. Hayles J. Baker S. Basham D. Bowman S. Brooks K. Brown D. Brown S. Chillingworth T. Churcher C. Collins M. Connor R. Cronin A. Davis P. Feltwell T. Fraser A. Gentles S. Goble A. Hamlin N. Harris D. Hidalgo J. Hodgson G. Holroyd S. Hornsby T. Howarth S. Huckle E.J. Hunt S. Jagels K. James K. Jones L. Jones M. Leather S. McDonald S. McLean J. Mooney P. Moule S. Mungall K. Murphy L. Niblett D. Odell C. Oliver K. O'Neil S. Pearson D. Quail M.A. Rabbinowitsch E. Rutherford K. Rutter S. Saunders D. Seeger K. Sharp S. Skelton J. Simmonds M. Squares R. Squares S. Stevens K. Taylor K. Taylor R.G. Tivey A. Walsh S. Warren T. Whitehead S. Woodward J. Volckaert G. Aert R. Robben J. Grymonprez B. Weltjens I. Vanstreels E. Rieger M. Schafer M. Muller-Auer S. Gabel C. Fuchs M. Dusterhoft A. Fritzc C. Holzer E. Moestl D. Hilbert H. Borzym K. Langer I. Beck A. Lehrach H. Reinhardt R. Pohl T.M. Eger P. Zimmermann W. Wedler H. Wambutt R. Purnelle B. Goffeau A. Cadieu E. Dreano S. Gloux S. Lelaure V. Mottier S. Galibert F. Aves S.J. Xiang Z. Hunt C. Moore K. Hurst S.M. Lucas M. Rochet M. Gaillardin C. Tallada V.A. Garzon A. Thode G. Daga R.R. Cruzado L. Jimenez J. Sanchez M. del Rey F. Benito J. Dominguez A. Revuelta J.L. Moreno S. Armstrong J. Forsburg S.L. Cerutti L. Lowe T. McCombie W.R. Paulsen I. Potashkin J. Shpakovski G.V. Ussery D. Barrell B.G. Nurse P. Nature. 2002; 415: 871-880Crossref PubMed Scopus (1251) Google Scholar), officially putting S. pombe into the post-genomic era. Although S. pombe is a well studied model eukaryote, experimental resources and information for functional genomics are so far limited. Recently, we established a proteome resource by expressing the ORFeome 4The abbreviations used are: ORF, open reading frame; WB, Western blotting; GRAVY, grand average hydrophobicity; ER, endoplasmic reticulum; HA, hemagglutinin; MM, minimal medium; EF, elongation factor; eIF, eukaryotic translation initiation factor. of S. pombe at moderate to high levels under the control of the inducible promoter nmt1 (7Matsuyama A. Arai R. Yashiroda Y. Shirai A. Kamata A. Sekido S. Kobayashi Y. Hashimoto A. Hamamoto M. Hiraoka Y. Horinouchi S. Yoshida M. Nat. Biotechnol. 2006; 24: 841-847Crossref PubMed Scopus (454) Google Scholar). In the present study, we report electrophoresis-based analyses of the fission yeast proteome, including the collection of information regarding gel mobility of the proteome. This unique data set enables one to identify an unknown target protein detected on a gel, even if it is hard to identify by classical purification-based methods. Furthermore, we identified several novel acetylated or methylated proteins, using a global screen based on induced expression of the tagged proteins in combination with Western blotting (WB). S. pombe Strains and Media—S. pombe strains used in this study are listed in supplemental Table S1. A series of S. pombe strains in which each ORF is fused with two FLAG epitopes and a hexahistidine tag was integrated at the leu1 locus were derived from AM2 (7Matsuyama A. Arai R. Yashiroda Y. Shirai A. Kamata A. Sekido S. Kobayashi Y. Hashimoto A. Hamamoto M. Hiraoka Y. Horinouchi S. Yoshida M. Nat. Biotechnol. 2006; 24: 841-847Crossref PubMed Scopus (454) Google Scholar). The construction of these strains is described elsewhere (7Matsuyama A. Arai R. Yashiroda Y. Shirai A. Kamata A. Sekido S. Kobayashi Y. Hashimoto A. Hamamoto M. Hiraoka Y. Horinouchi S. Yoshida M. Nat. Biotechnol. 2006; 24: 841-847Crossref PubMed Scopus (454) Google Scholar). AM5 was used for multicopy overexpression of an ORF. Complete medium YE (0.5% yeast extract, 2% glucose, 5 μg/ml adenine) was used for routine culture of S. pombe strains. Selective minimal medium SD (8Sherman F. Fink G. Hick J. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1986Google Scholar) containing only 1% glucose was used for routine culture of S. pombe transformants. Minimal medium MM (9Moreno S. Klar A. Nurse P. Methods Enzymol. 1991; 194: 795-823Crossref PubMed Scopus (3148) Google Scholar) was used for expression of genes driven by the nmt1 promoter (10Maundrell K. J. Biol. Chem. 1990; 265: 10857-10864Abstract Full Text PDF PubMed Google Scholar). Adenine, leucine, and uracil were added to the medium (final concentration, 50 μg/ml) when needed. General Methods and Transformation of S. pombe—General methods to handle fission yeast cells were as described (11Gutz, H., Heslot, H., Leupold, U., and Loprieno, N. (1974) in Handbook of Genetics (King, R., ed) pp. 395-446, Plenum Publishing Corp., New YorkGoogle Scholar). A high efficiency protocol for transformation of S. pombe cells, as described previously (12Matsuyama A. Yabana N. Watanabe Y. Yamamoto M. Genetics. 2000; 155: PubMed Google Scholar), was modified to the The cells were in medium and with and in of was added as into each of A. Shirai A. Yashiroda Y. Kamata A. Horinouchi S. Yoshida M. PubMed Scopus Google Scholar) and its each was with or in a of and of the was used with of 40% was added to the and the was at for at the addition of of the cells were at for in and on the SD the cells a or were on the SD and of were subsequently on the MM and for at The cells were and used for the of Gel of in were by the method described by V. V. 2000; PubMed Scopus Google Scholar) with modification R., and M. Nat. Scholar). of tagged strains on MM for at were in of MM medium and in of were added into the The cells were for 5 at in of SDS gel buffer, and for 5 of of the were on a polyacrylamide gel The proteins were to and for with 2% in with were A tag and or a with was as a with the The was with and for with a an with with the proteins were detected using the system As a a protein containing a of all and was in each gel. of gel mobility of proteins in we the gel mobility of each band of the by an tag and its molecular mass in each and calculated the for each The molecular masses of all proteins in fission yeast were from gel mobility on SDS-PAGE according to these The gel mobility of each was using one band was detected in WB, was with a multicopy were used only when was in using in This the tag and the were from The was from The an acetylated lysine and a methylated lysine and were described elsewhere Y. Y. Yoshida M. J. PubMed Scopus Google Scholar, A. T. Y. A. Y. D. H. Y. N. S. Horinouchi S. Yoshida M. J. 2002; PubMed Scopus Google Scholar, H. Yoshida M. Y. PubMed Scopus Google Scholar). and sequence information was from the S. pombe data The presence of was calculated by using Biol. PubMed Scopus Google Scholar). by of proteins was according to the analysis of (7Matsuyama A. Arai R. Yashiroda Y. Shirai A. Kamata A. Sekido S. Kobayashi Y. Hashimoto A. Hamamoto M. Hiraoka Y. Horinouchi S. Yoshida M. Nat. Biotechnol. 2006; 24: 841-847Crossref PubMed Scopus (454) Google Scholar). was used for calculated using the for the gel mobility and the or of proteins, as well as the for the gel mobility of proteins and their The were for by the Meanwhile, the the calculated and molecular masses was by the gel mobility and basic data of proteins is on our are to and the data The an on Gel of set a for global analysis of gel mobility of proteins in we S. pombe in which each of the fused with two of the FLAG and tag at the is integrated at the leu1 locus A. Shirai A. Yashiroda Y. Kamata A. Horinouchi S. Yoshida M. PubMed Scopus Google Scholar) This collection was established based on a ORFeome resource that more than of genes by the genome (7Matsuyama A. Arai R. Yashiroda Y. Shirai A. Kamata A. Sekido S. Kobayashi Y. Hashimoto A. Hamamoto M. Hiraoka Y. Horinouchi S. Yoshida M. Nat. Biotechnol. 2006; 24: 841-847Crossref PubMed Scopus (454) Google Scholar). were under the nmt1 promoter K. PubMed Scopus Google Scholar). in cells in MM, was separated by SDS-PAGE using a gel. Gel mobility was by WB, using an tag or an In the was we the from multicopy to A. Shirai A. Yashiroda Y. Kamata A. Horinouchi S. Yoshida M. PubMed Scopus Google Scholar). we have the gel mobility of proteins, to 93.4% of all in the genome Table the “mobilitome,” a novel data set of gel mobility of the proteome. The mobilitome not only the gel of of proteins of when The size of proteins by gel mobility was not from that of proteins calculated from their as by the the were one by however, more than 40% of the did not migrate of the each ORF size for the and was a the calculated molecular mass and the molecular mass as by the This that it is difficult to gel mobility of proteins in SDS-PAGE from their Gel mobility is by properties of proteins. the hydrophobicity by the J. J. Biol. PubMed Scopus Google Scholar) had the on gel Statistical analysis revealed that proteins to gel mobility of a protein became as its proteins with mobility to have a high with mobility have a low This is to the in which SDS to proteins. In SDS to a protein at a of one R. Biochem. J. PubMed Scopus Google Scholar, C. J. Biol. Chem. 1970; Full Text PDF PubMed Google Scholar). However, because this is the SDS to the of proteins C. PubMed Scopus Google Scholar). As a it is that proteins of SDS than and migrate in Furthermore, the of proteins gel mobility to proteins for which are than mobility than calculated this be to with Although proteins showed these physicochemical properties on the were a of proteins that did not this was a of proteins with mobility than It that these proteins are subject to post-translational modifications that their of proteins that have so far been to be in fission proteins showed mobility than Although the two did not such the proteins were by which be derived from not In addition to these the molecular or functional characteristics of proteins such as to have a on gel proteins in with the most proteins were to migrate more than In proteins in the endoplasmic or the showed a to migrate than calculated proteins for which gel mobility was hard to were proteins that at the or It that this could be to the hydrophobicity of the proteins and the post-translational modifications as proteins that are in the or the had a high in proteins are which to It is known that proteins have that are to Therefore, one would that the high mobility of proteins is to be by their than their However, analysis revealed that of gel of proteins was to that of proteins as by the proteins relatively with their these proteins are not as as and proteins. Furthermore, the of gel of proteins was to that of the proteins as by the The for this is still it that unknown protein or post-translational modification could be In addition to the of gel mobility of the we detected proteins of proteins with that than the showed that a a or a as well as of these of post-translational modifications such as and PubMed Scopus Google Scholar, Biochem. A PubMed Scopus Google Scholar, J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) not the to be that than the were in proteins not these have a in the it is difficult to from the The of the nmt1 promoter for expression could a of expression of proteins that are not in which could of proteins. Therefore, it that the high mobility of proteins is by their To this we the the presence of and gel mobility of the however, analysis revealed that proteins than proteins with such as by the supplemental the was that it is that the by expression is the for the mobility of proteins than Identification of the global mobility data we enables one to identify a protein on the gel. an unknown protein of detected by a for this protein can be using the based on the real of gel mobility, and the expressing tagged can be each one by one in the as the to the identification of the because the containing the target protein is to when one of which is the and the of which is the for which mobility is by the addition of the this we set a that for by using gel mobility data of target proteins. As a of we to identify an S. pombe protein detected by an tag This protein was as a band with a size of when the was with for for this in the mobilitome data and proteins. the of each by the as the mobilitome using the As a of the screen of we identified two that two the translation elongation a sequence in the sequence is for the by a The a sequence lost the to the that the sequence as the in Although it that a methodology could be to the sequence data it would not be because of the low and proteins that with of for only of the proteome, that only of could be a methodology were to be used for the identification of this protein based on sequence data and ORF at would to be because of the low and To this methodology to the identification of novel acetylated proteins, we for proteins for the two only in the of the an deacetylase B.J. E. Biol. PubMed Scopus Google Scholar), using an of these acetylated proteins were as and in using an A screen of from the mobilitome data with of in the identification of two proteins, and P. PubMed Google Scholar), which are of eukaryotic translation initiation and In this to identify and using the sequence data it would have been to for proteins of which to of the proteome Although of eIF5A has been previously in R. Y. Y. H. J. T. Y. H. L. M. Y. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, H. R. C. M. F. J. PubMed Scopus Google Scholar), the of However, the that eIF5A is by the deacetylase on the of protein for or of eIF5A in the screen for acetylated proteins us to screen for a of modified proteins. As in was detected with an in the was However, when they were of these proteins became even in cells that overexpression to of modified proteins that are not detected in the on this we all the proteins for acetylated or methylated proteins by SDS-PAGE by using and antibodies, which are a with or proteins Y. Y. Yoshida M. J. PubMed Scopus Google Scholar, H. Yoshida M. Y. PubMed Scopus Google Scholar). a were even when these were used the of cells As however, by tagged proteins one by we identified twelve and eight proteins the sequence were as that were to be acetylated or and supplemental Table The of these modified proteins the of the in using a protein known to be acetylated and methylated in fission was identified in this as a of In this proteins and as well as eukaryotic translation elongation and were identified as proteins, that is in protein to have relatively in proteins are methylated in S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S.J. S. L. Y. R. Acad. Sci. S. A. 2002; PubMed Scopus Google Scholar, J. J. Biochem. PubMed Scopus Google Scholar, T. A. Biochem. J. PubMed Scopus Google Scholar), and lysine of has been widely J. W. K. Biophys. PubMed Scopus Google Scholar). Although of the fission yeast had not been known, our demonstrated that this is the in fission the of has not been so far in any by gel mobility of and proteins and are detected in To these proteins are for we genes that as well as the which a of the yeast for lysine of H. H. P. Y. K. 2002; PubMed Scopus Google Scholar) Western analysis of showed that in the and the of and the protein was that the proteins are in of proteins as well as This was by expressing the tagged or the protein in the Recently, a yeast of was to the protein S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, the regulation of this protein by be the two It has been that the protein is methylated by S. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar) in S. which is a of However, of and the protein was not in these including the that are in of these In this we an electrophoresis-based analysis of the fission yeast proteome, including of the mobilitome and a screen for acetylated or methylated proteins. The mobilitome data set showed two more than 40% of the proteins in S. pombe did not migrate to the from their it was that most proteins at the calculated in the yeast M.A. Y. N. L. S. H. M. M. E.J. PubMed Scopus Google Scholar). SDS-PAGE is the most widely used method for determination of the molecular size of in a However, SDS to the of proteins and to a protein with according to its which with the analyses used in this study revealed that gel mobility of proteins was by the hydrophobicity and of proteins. Furthermore, of the proteome had the post-translational modifications that gel modifications and modifications. The data set of gel mobility of the fission yeast proteome provides not only useful information on physicochemical properties for functional of such is the of the mobilitome for the systematic identification of a target protein of detected on a gel. Despite its simplicity, this methodology has for the identification of target proteins. example, because and used in this study high sensitivity in not have to or proteins H. Yoshida M. Y. PubMed Scopus Google Scholar), it was to purification-based In because most post-translational in such as in only a of the cells, the of the target modifications is sometimes for identification by classical methods. However, the with its high coverage can these because it only of the target and not on its In any protein we were to detect on a gel with a can be identified using the the protein is not listed in the mobilitome data The high coverage more than of the proteome, the of this In we have information in the data on the of more in addition to the of proteins, which sometimes the gel mobility, the of the target using protein data collection of band information identify such modified proteins by the To post-translational modifications have been described K. W. Enzymol. Biol. Google Scholar), which the of an of global that be the the of the post-translational modifications the a using mass identified a large of proteins in cells R. Y. Y. H. J. T. Y. H. L. M. Y. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). In this study, we identified methylated and acetylated proteins using a reverse and most of the proteins were not known to be subject to such the modifications. however, all the acetylated proteins would be identified they were because of proteins than is not in of of It is still that overexpression could the be with an screen for modified proteins as the step to the of a modification of Thus, in combination with the reverse proteomic the mobilitome and the methodology described as a to the of post-translational modifications. at the of the for for useful in with