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... energy was used to promote fragmentation, typical average values for doubly charged ions were 41 and 56 V for m/z 600 and 900, respectively, and for triply charged ions typical average values were 29 and 43 V for m/z 600 and 900, respectively. The collision energy range was 20% higher than that used for unlabeled peptides to overcome the stabilizing effect of the basic N-terminal derivative and achieve equivalent fragmentation. Data Analysis and Interpretation—Peptide and protein identifications were performed using the Mascot search engine (ver. 1.9; Matrix Science, London, United Kingdom) (12). Database searching was restricted to tryptic peptides of yeast (Swiss-Prot version 42.5; 4,924 Saccharomyces cerevisiae sequences; 138,922 total sequences). Sacetamido, N-terminal, and lysine modifications were selected as fixed, methionine oxidation as variable, one missed cleavage allowed and precursor error tolerance at 50 ppm. Full trypsin specificity (Nand C-terminal was also applied. Signature-ion peak areas from the isobaric tags were extracted from the 4700 or QSTAR raw data and matched to identified peptides using prototype software tools. The complete list of identified peptides w...

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... measurement of expression levels of relevant sets of proteins. Recently, quantitative approaches utilizing MS and a host of stable isotope-labeling chemistries have emerged (reviewed in Refs. 1 and 2), offering a departure from traditional techniques employing comparative two-dimensional gel electrophoresis. The ICAT quantitative labeling strategy (3, 4) is perhaps the best-characterized method for relative protein quantitation using MS. Other elegant approaches use cell-culture enrichment with a stable isotope-labeled amino acid, including arginine (5), lysine (6), tyrosine (7), and leucine (8), for in vivo incorporation of a mass difference to support relative quantitation. This circumvents potential difficulties surrounding chemical labeling downstream in a comparative experiment. All of these methods impart a mass difference as the basis for quantitation by measurement of relative peak areas of MS and/or MS/MS mass spectra. There are, however, a number of limitations imposed by mass-difference labeling. The mass-difference concept for many practical purposes is limited to a binary (2-plex) set of reagents, and this makes comparison of multiple states (e.g. several experimental co...

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...nd that inactivation of Upf1p and Xrn1p causes common as well as unique effects on protein expression. Molecular & Cellular Proteomics 3:1154–1169, 2004. An initial step in the systematic investigation of cellular processes is the identification and measurement of expression levels of relevant sets of proteins. Recently, quantitative approaches utilizing MS and a host of stable isotope-labeling chemistries have emerged (reviewed in Refs. 1 and 2), offering a departure from traditional techniques employing comparative two-dimensional gel electrophoresis. The ICAT quantitative labeling strategy (3, 4) is perhaps the best-characterized method for relative protein quantitation using MS. Other elegant approaches use cell-culture enrichment with a stable isotope-labeled amino acid, including arginine (5), lysine (6), tyrosine (7), and leucine (8), for in vivo incorporation of a mass difference to support relative quantitation. This circumvents potential difficulties surrounding chemical labeling downstream in a comparative experiment. All of these methods impart a mass difference as the basis for quantitation by measurement of relative peak areas of MS and/or MS/MS mass spectra. There are, how...

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...protein. Proteins Up-regulated in the upf1 and xrn1 Strains—The data clearly indicate that deletion of the Xrn1 and Upf1 proteins produces similar changes to the protein phenotype (Fig. 6). Of the 48 proteins considered to be up-regulated in the xrn1 strain, 23 are seen to be also up-regulated in the upf1 strain. The magnitude of increases is, however, two to three times greater in the xrn1 strain. Similarity in protein upregulation also extends to ontological comparison of these two mutant strains (Fig. 6). Up-regulated proteins were grouped according to biological function as described (18). Significant groups were chosen by comparison with those identified proteins that were not significantly up- or downregulated (p 0.05). Closer inspection reveals that many of the proteins are involved in amino acid biosynthesis (including many aspects of amine and nitrogen metabolism). Enzymes involved in the biosynthesis of each of the 20 common amino acids as well as enzymes involved in general nitrogen and amine metabolism (e.g. urea cycle and general metabolism of amine groups) are up-regulated in both xrn1 and upf1 strains. This confirms the findings of the earlier ICAT study compari...

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...nificant changes in levels in upf1 cells, there is no significant correlation between mRNA and protein levels (r 0.19). Likewise, in xrn1 cells, proteins showing significant increases or decreases in levels also show no significant correlation with the levels of their respective mRNAs (Fig. 7B; r 0.20). These comparisons indicate that the levels of a small number of proteins in the upf1 and xrn1 strains are regulated post-transcriptionally, i.e. for some proteins there are substantive differences between the respective changes in mRNA and protein levels. In agreement with previous studies (34, 35), these measurements of protein and mRNA levels are revealing quite different and nonoverlapping aspects of the overall phenotypic changes induced by the deletion of these factors. CONCLUSIONS We have used a multiplexed peptide quantitation methodology to identify global protein expression trends in a set of isogenic yeast strains. This approach made use of a set of four isobaric peptide derivatization reagents that yield informative MS/MS spectra for peptide identification and quantitation. The derivatized peptides are indistinguishable by their MS spectra or MS/MS ion series, but exhibit int...

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...nd that inactivation of Upf1p and Xrn1p causes common as well as unique effects on protein expression. Molecular & Cellular Proteomics 3:1154–1169, 2004. An initial step in the systematic investigation of cellular processes is the identification and measurement of expression levels of relevant sets of proteins. Recently, quantitative approaches utilizing MS and a host of stable isotope-labeling chemistries have emerged (reviewed in Refs. 1 and 2), offering a departure from traditional techniques employing comparative two-dimensional gel electrophoresis. The ICAT quantitative labeling strategy (3, 4) is perhaps the best-characterized method for relative protein quantitation using MS. Other elegant approaches use cell-culture enrichment with a stable isotope-labeled amino acid, including arginine (5), lysine (6), tyrosine (7), and leucine (8), for in vivo incorporation of a mass difference to support relative quantitation. This circumvents potential difficulties surrounding chemical labeling downstream in a comparative experiment. All of these methods impart a mass difference as the basis for quantitation by measurement of relative peak areas of MS and/or MS/MS mass spectra. There are, how...

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... automatically during the course of the experiment, and the intensity of the synthetic peptide-derived signature ion at m/z 117.1 was used to calculate an absolute value. We can estimate using the molecular weight (135,417) of carbamoyl phosphate synthetase that there were 26 ng of this protein in the original 150 g of wild-type yeast lysate. This calculates to 45,000 copies per cell in the wild-type strain, rising to 98,000 copies/cell in xrn1. This approach, where added internal peptide standards remain isobaric, is significantly different from the absolute quantitation (AQUA) approach (17), where a mass-difference approach is employed through isotopic enrichment of the synthetic peptides. Finally, we were also able to perform a more specific analysis to confirm the deletion of the Xrn1 protein in the xrn1 yeast strain (Fig. 5B), 2 Pilot studies were conducted as a 2-plex experiment using 100 g of total protein with similar conditions for protein extraction, digestion, labeling, chromatography, and MS. TABLE I—continued Protein name Counta Xrn1 ratiob Xrn1 S.D. Upf1 ratiob Upf1 S.D. Xrn1 fold changec Repd,e (P32591) Transcription regulatory protein SWI3 (SWI/SNF complex c...

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...eb1 (29), a sequence-specific DNA-binding protein that recognizes sites within both the enhancer and the promoter regions of the rRNA genes as well as sites upstream of many genes transcribed by RNA polymerase II. Also downregulated are the Trm1 and METL (S-adenosyl methionine sythetase) proteins, which are required for methylation of cellular tRNAs (30). While the basis for these observations is unclear, it is possible that the down-regulation of proteins involved in protein translation in xrn1 cells reflects a regulatory circuit responding to the loss of Xrn1p’s role in pre-rRNA processing (31, 32) and that the up-regulation of proteins involved in amino acid biosynthesis in both mutant strains may occur in these strains as a consequence of restoring functional translation of several endogenous nonsense-containing mRNAs encoding enzymes in histidine, arginine, and leucine biosynthesis (33). Relative Changes in mRNA and Protein Levels—He et al. (19) have previously used high-density oligonucleotide microarray analysis to assess global changes in mRNA abundance in the upf1 and xrn1 strains. We have utilized this data to determine whether there is a correlation between the relative chang...

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...VTGLVR (derivatized m/z 2015.1) that corresponds to residues 861–878 of the native Xrn1 protein. This peptide (and hence protein) is clearly absent only in the xrn1 strain. Multiplexed Quantitation Using Isobaric Reagents 1164 Molecular & Cellular Proteomics 3.12 at C am bridge U niversity Library on O ctober 16, 2007 w w w .m cponline.org D ow nloaded from teins also yields distinct protein phenotypes. Previous xrn1 and upf1 deletion or mutation studies have shown that removal or inhibition of the activities of these two proteins resulted in significantly increased levels of cellular mRNA (19). One pathway of mRNA decay in yeast involves poly(A) shortening followed by decapping and then 5 to 3 decay. Cells devoid of Xrn1p therefore accumulate mRNAs that have shortened or no poly(A) tails and which may lack the 5 cap structure (20). We find that the xrn1 strain showed specific up-regulation of the ribosome biogenesis protein BMS1 required for the maturation of the 40S ribosomal subunit (21), the SC24 protein required to promote the transport of secretory, membrane, and vacuolar proteins from the endoplasmic reticulum to the Golgi complex (22), and the RNA polymerase II transcrip...

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...nificant changes in levels in upf1 cells, there is no significant correlation between mRNA and protein levels (r 0.19). Likewise, in xrn1 cells, proteins showing significant increases or decreases in levels also show no significant correlation with the levels of their respective mRNAs (Fig. 7B; r 0.20). These comparisons indicate that the levels of a small number of proteins in the upf1 and xrn1 strains are regulated post-transcriptionally, i.e. for some proteins there are substantive differences between the respective changes in mRNA and protein levels. In agreement with previous studies (34, 35), these measurements of protein and mRNA levels are revealing quite different and nonoverlapping aspects of the overall phenotypic changes induced by the deletion of these factors. CONCLUSIONS We have used a multiplexed peptide quantitation methodology to identify global protein expression trends in a set of isogenic yeast strains. This approach made use of a set of four isobaric peptide derivatization reagents that yield informative MS/MS spectra for peptide identification and quantitation. The derivatized peptides are indistinguishable by their MS spectra or MS/MS ion series, but exhibit int...

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...led residue. The sensitivity enhancement is carried over into MS/MS spectra, because all of the peptide backbone fragments ions are also isobaric (Fig. 2). One potential drawback of this approach is that MS/MS spectra must be acquired, which requires more analysis time than performing result-dependent analysis only on differentially expressed peptide pairs in MS (e.g. with ICAT). We feel, however, that the ability to identify more proteins with increased confidence and greater peptide coverage outweighs this disadvantage. Strategies have been described that employ isobaric peptide derivatives (13). To date, reaction of these reagents with complex peptide mixtures has not been shown. The use of a discreet, highly abundant, low-mass MS/MS signature ion as described in this work provides an unambiguous coding system without introducing additional sources of complexity into the mass spectrum. Finally, we use tags that generate abundant signature ions under MS/MS conditions optimal for peptide fragmentation. The reagents described here contain N-methylpiperazine, a moderately strong base that conveys useful properties to the FIG. 2. Example MS/MS spectrum of peptide TPHPALTEAK from a protei...

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..., export, translation, and turnover (26), the initiating factor eIF-5A (which may have a role in regulating exonucleolytic decay (27)), and the elongation factor-1 subunit. In contrast, many of the down-regulated proteins in the upf1 strain are involved in DNA replication or RNA transcription. Approximately one-third of the proteins down-regulated in upf1 cells are involved in chromosome and chromatin structure (histones) or transcriptional regulation. These include the Swi3 protein, which is a global activator of transcription required for the induced expression of a large number of genes (28), and Reb1 (29), a sequence-specific DNA-binding protein that recognizes sites within both the enhancer and the promoter regions of the rRNA genes as well as sites upstream of many genes transcribed by RNA polymerase II. Also downregulated are the Trm1 and METL (S-adenosyl methionine sythetase) proteins, which are required for methylation of cellular tRNAs (30). While the basis for these observations is unclear, it is possible that the down-regulation of proteins involved in protein translation in xrn1 cells reflects a regulatory circuit responding to the loss of Xrn1p’s role in pre-rRNA proc...

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.... Of particular interest is validation of quantitation via a peptide-based workflow whereby protein extraction, digestion, and labeling are performed in parallel, prior to mixing labeled samples for chromatography and MS. A well-characterized system such as yeast provides the opportunity to validate some novel aspects of this quantitative methodology. In this study, we have examined the global protein expression of a wild-type yeast strain and the isogenic upf1 and xrn1 mutant strains that are defective in the nonsense-mediated mRNA decay and the general 5 to 3 decay pathway, respectively (9, 10). A variety of global changes are observed, including consistent up-regulation of a common set of proteins involved in amino acid biosynthetic pathways in both upf1 and xrn1 strains, and specific down-regulation of proteins of the translation apparatus in the xrn1 strain. MATERIALS AND METHODS Protein Mixture—A protein mix consisting of equimolar amounts of BSA, -casein, alcohol dehydrogenase, lysozyme, -galactosidase, and serotransferrin (all from Sigma, Milwaukee, WI) was reduced (2 mM Tris-(2-carboxyethyl)phosphine (TCEP), 37 °C, 1 h), alkylated (5 mM iodoacetamide, 37 °C, 2 h) and dig...

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...ellular processes is the identification and measurement of expression levels of relevant sets of proteins. Recently, quantitative approaches utilizing MS and a host of stable isotope-labeling chemistries have emerged (reviewed in Refs. 1 and 2), offering a departure from traditional techniques employing comparative two-dimensional gel electrophoresis. The ICAT quantitative labeling strategy (3, 4) is perhaps the best-characterized method for relative protein quantitation using MS. Other elegant approaches use cell-culture enrichment with a stable isotope-labeled amino acid, including arginine (5), lysine (6), tyrosine (7), and leucine (8), for in vivo incorporation of a mass difference to support relative quantitation. This circumvents potential difficulties surrounding chemical labeling downstream in a comparative experiment. All of these methods impart a mass difference as the basis for quantitation by measurement of relative peak areas of MS and/or MS/MS mass spectra. There are, however, a number of limitations imposed by mass-difference labeling. The mass-difference concept for many practical purposes is limited to a binary (2-plex) set of reagents, and this makes comparison of mu...

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...re substantially different (Table I, Fig. 6). More than half of the proteins down-regulated in xrn1 cells are structural components of the ribosome or are factors involved in protein synthesis. These include the poly(A)-binding protein Pab1p that FIG. 5—continued Multiplexed Quantitation Using Isobaric Reagents Molecular & Cellular Proteomics 3.12 1165 at C am bridge U niversity Library on O ctober 16, 2007 w w w .m cponline.org D ow nloaded from also serves as a scaffold for a series of post-transcriptional regulatory factors involved in mRNA 3 processing, export, translation, and turnover (26), the initiating factor eIF-5A (which may have a role in regulating exonucleolytic decay (27)), and the elongation factor-1 subunit. In contrast, many of the down-regulated proteins in the upf1 strain are involved in DNA replication or RNA transcription. Approximately one-third of the proteins down-regulated in upf1 cells are involved in chromosome and chromatin structure (histones) or transcriptional regulation. These include the Swi3 protein, which is a global activator of transcription required for the induced expression of a large number of genes (28), and Reb1 (29), a sequence-specific...

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...2.6.1.42) 6 0.521 0.073 0.522 0.027 1.165 Yes (Q08965) Ribosome biogenesis protein BMS1 2 0.625 0.016 0.521 0.014 1.159 Upf1-down (Q03667) Hypothetical 16.7-kDa protein in CDC5-MVP1 intergenic region 2 0.469 0.18 0.344 0.084 0.549 Yes (P43579) Hypothetical 78.8-kDa protein in HSP12-HXT10 intergenic region 2 0.577 0.027 0.376 0.032 0.634 (P04912) Histone H2A.2 2 0.42 0.005 0.379 0.051 0.642 (P87108) Mitochondrial import inner membrane translocase subunit TIM10 2 0.502 0.044 0.41 0.012 0.732 Yes (P38693) Acid phosphatase PHO12 precursor (EC 3.1.3.2) 4 0.414 0.098 0.416 0.05 0.751 Yes (P15565) N(2),N(2)-dimethylguanosine tRNA methyltransferase, mitochondrial prec. 2 0.516 0.125 0.417 0.005 0.753 Yes Multiplexed Quantitation Using Isobaric Reagents 1162 Molecular & Cellular Proteomics 3.12 at C am bridge U niversity Library on O ctober 16, 2007 w w w .m cponline.org D ow nloaded from (Table I). We compared the current set of differentially expressed proteins with relative expression data from a 2-plex pilot scale study2 of a separate batch of protein lysates from the wild-type and xrn1 strains. This comparison revealed that 86% of up-regulated and 79% of down-regulated proteins (Table I...

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.... Of particular interest is validation of quantitation via a peptide-based workflow whereby protein extraction, digestion, and labeling are performed in parallel, prior to mixing labeled samples for chromatography and MS. A well-characterized system such as yeast provides the opportunity to validate some novel aspects of this quantitative methodology. In this study, we have examined the global protein expression of a wild-type yeast strain and the isogenic upf1 and xrn1 mutant strains that are defective in the nonsense-mediated mRNA decay and the general 5 to 3 decay pathway, respectively (9, 10). A variety of global changes are observed, including consistent up-regulation of a common set of proteins involved in amino acid biosynthetic pathways in both upf1 and xrn1 strains, and specific down-regulation of proteins of the translation apparatus in the xrn1 strain. MATERIALS AND METHODS Protein Mixture—A protein mix consisting of equimolar amounts of BSA, -casein, alcohol dehydrogenase, lysozyme, -galactosidase, and serotransferrin (all from Sigma, Milwaukee, WI) was reduced (2 mM Tris-(2-carboxyethyl)phosphine (TCEP), 37 °C, 1 h), alkylated (5 mM iodoacetamide, 37 °C, 2 h) and dig...

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...lar Proteomics 3.12 at C am bridge U niversity Library on O ctober 16, 2007 w w w .m cponline.org D ow nloaded from teins also yields distinct protein phenotypes. Previous xrn1 and upf1 deletion or mutation studies have shown that removal or inhibition of the activities of these two proteins resulted in significantly increased levels of cellular mRNA (19). One pathway of mRNA decay in yeast involves poly(A) shortening followed by decapping and then 5 to 3 decay. Cells devoid of Xrn1p therefore accumulate mRNAs that have shortened or no poly(A) tails and which may lack the 5 cap structure (20). We find that the xrn1 strain showed specific up-regulation of the ribosome biogenesis protein BMS1 required for the maturation of the 40S ribosomal subunit (21), the SC24 protein required to promote the transport of secretory, membrane, and vacuolar proteins from the endoplasmic reticulum to the Golgi complex (22), and the RNA polymerase II transcription elongation factor TFS2 required for efficient transcription elongation past template-encoded arresting sites. The upf1 strain showed up-regulation of proteins such as the Sec63 protein important for protein assembly in the nucleus and endo...

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... xrn1 strain showed specific up-regulation of the ribosome biogenesis protein BMS1 required for the maturation of the 40S ribosomal subunit (21), the SC24 protein required to promote the transport of secretory, membrane, and vacuolar proteins from the endoplasmic reticulum to the Golgi complex (22), and the RNA polymerase II transcription elongation factor TFS2 required for efficient transcription elongation past template-encoded arresting sites. The upf1 strain showed up-regulation of proteins such as the Sec63 protein important for protein assembly in the nucleus and endoplasmic reticulum (23), the Sis1 protein required for normal initiation of translation (24), the polymerase II transcription factor TFIIF that promotes transcriptional elongation (25), and the t-complex protein 1 that acts as a molecular chaperone for protein folding. Proteins Down-regulated in the xrn1 and upf1 Strains— In contrast to the general similarity of proteins up-regulated in the xrn1 and upf1 strains, those proteins that are significantly down-regulated appear to be quite different. Of the 39 proteins down-regulated in the xrn1 strain, only three are common to the upf1 strain. Ontological classific...

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...Previous xrn1 and upf1 deletion or mutation studies have shown that removal or inhibition of the activities of these two proteins resulted in significantly increased levels of cellular mRNA (19). One pathway of mRNA decay in yeast involves poly(A) shortening followed by decapping and then 5 to 3 decay. Cells devoid of Xrn1p therefore accumulate mRNAs that have shortened or no poly(A) tails and which may lack the 5 cap structure (20). We find that the xrn1 strain showed specific up-regulation of the ribosome biogenesis protein BMS1 required for the maturation of the 40S ribosomal subunit (21), the SC24 protein required to promote the transport of secretory, membrane, and vacuolar proteins from the endoplasmic reticulum to the Golgi complex (22), and the RNA polymerase II transcription elongation factor TFS2 required for efficient transcription elongation past template-encoded arresting sites. The upf1 strain showed up-regulation of proteins such as the Sec63 protein important for protein assembly in the nucleus and endoplasmic reticulum (23), the Sis1 protein required for normal initiation of translation (24), the polymerase II transcription factor TFIIF that promotes transcripti...

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...ich are required for methylation of cellular tRNAs (30). While the basis for these observations is unclear, it is possible that the down-regulation of proteins involved in protein translation in xrn1 cells reflects a regulatory circuit responding to the loss of Xrn1p’s role in pre-rRNA processing (31, 32) and that the up-regulation of proteins involved in amino acid biosynthesis in both mutant strains may occur in these strains as a consequence of restoring functional translation of several endogenous nonsense-containing mRNAs encoding enzymes in histidine, arginine, and leucine biosynthesis (33). Relative Changes in mRNA and Protein Levels—He et al. (19) have previously used high-density oligonucleotide microarray analysis to assess global changes in mRNA abundance in the upf1 and xrn1 strains. We have utilized this data to determine whether there is a correlation between the relative changes in protein and mRNA levels in the upf1 and xrn1 strains. Fig. 7 compares average fold changes in RNA abundance (from four independent experiments) to the relaFIG. 6. Display of ontology profile from an ontology search (18) of significant up- and down-regulated proteins observed in the multip...

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...dentification and measurement of expression levels of relevant sets of proteins. Recently, quantitative approaches utilizing MS and a host of stable isotope-labeling chemistries have emerged (reviewed in Refs. 1 and 2), offering a departure from traditional techniques employing comparative two-dimensional gel electrophoresis. The ICAT quantitative labeling strategy (3, 4) is perhaps the best-characterized method for relative protein quantitation using MS. Other elegant approaches use cell-culture enrichment with a stable isotope-labeled amino acid, including arginine (5), lysine (6), tyrosine (7), and leucine (8), for in vivo incorporation of a mass difference to support relative quantitation. This circumvents potential difficulties surrounding chemical labeling downstream in a comparative experiment. All of these methods impart a mass difference as the basis for quantitation by measurement of relative peak areas of MS and/or MS/MS mass spectra. There are, however, a number of limitations imposed by mass-difference labeling. The mass-difference concept for many practical purposes is limited to a binary (2-plex) set of reagents, and this makes comparison of multiple states (e.g. severa...

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...s protein BMS1 required for the maturation of the 40S ribosomal subunit (21), the SC24 protein required to promote the transport of secretory, membrane, and vacuolar proteins from the endoplasmic reticulum to the Golgi complex (22), and the RNA polymerase II transcription elongation factor TFS2 required for efficient transcription elongation past template-encoded arresting sites. The upf1 strain showed up-regulation of proteins such as the Sec63 protein important for protein assembly in the nucleus and endoplasmic reticulum (23), the Sis1 protein required for normal initiation of translation (24), the polymerase II transcription factor TFIIF that promotes transcriptional elongation (25), and the t-complex protein 1 that acts as a molecular chaperone for protein folding. Proteins Down-regulated in the xrn1 and upf1 Strains— In contrast to the general similarity of proteins up-regulated in the xrn1 and upf1 strains, those proteins that are significantly down-regulated appear to be quite different. Of the 39 proteins down-regulated in the xrn1 strain, only three are common to the upf1 strain. Ontological classification (18) also indicates that the effects of these two mutant strain...

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...lation, and turnover (26), the initiating factor eIF-5A (which may have a role in regulating exonucleolytic decay (27)), and the elongation factor-1 subunit. In contrast, many of the down-regulated proteins in the upf1 strain are involved in DNA replication or RNA transcription. Approximately one-third of the proteins down-regulated in upf1 cells are involved in chromosome and chromatin structure (histones) or transcriptional regulation. These include the Swi3 protein, which is a global activator of transcription required for the induced expression of a large number of genes (28), and Reb1 (29), a sequence-specific DNA-binding protein that recognizes sites within both the enhancer and the promoter regions of the rRNA genes as well as sites upstream of many genes transcribed by RNA polymerase II. Also downregulated are the Trm1 and METL (S-adenosyl methionine sythetase) proteins, which are required for methylation of cellular tRNAs (30). While the basis for these observations is unclear, it is possible that the down-regulation of proteins involved in protein translation in xrn1 cells reflects a regulatory circuit responding to the loss of Xrn1p’s role in pre-rRNA processing (31, 32)...

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...f the tag set, but remain isobaric. The example shown is the spectrum obtained from the singly charged [MH] peptide using a 4700 MALDI TOF-TOF analyzer, but the same holds true for any multiply charged peptide analyzed with an ESI-source mass spectrometer. Multiplexed Quantitation Using Isobaric Reagents Molecular & Cellular Proteomics 3.12 1157 at C am bridge U niversity Library on O ctober 16, 2007 w w w .m cponline.org D ow nloaded from tagged peptides. The use of cyclic amines as N-terminal peptide derivatives to simplify the interpretation of MS/MS spectra has been described previously (14,15). We find that the tags behave similarly in both MALDI and ESI, with a tendency to form more abundant and complete b- and y-ion series, while also reducing the proportion of ion current going into less informative fragmentation pathways. Our findings with the six-protein digest mixture show that the derivatization reaction itself is quite straightforward, requiring a simple room temperature reaction of 30 min. Residual reagent is easily quenched by the addition of one or more volumes of water prior to mixing, as decreasing the organic (ethanol) concentration accelerates hydrolysis of residual...

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...n xrn1 cells are structural components of the ribosome or are factors involved in protein synthesis. These include the poly(A)-binding protein Pab1p that FIG. 5—continued Multiplexed Quantitation Using Isobaric Reagents Molecular & Cellular Proteomics 3.12 1165 at C am bridge U niversity Library on O ctober 16, 2007 w w w .m cponline.org D ow nloaded from also serves as a scaffold for a series of post-transcriptional regulatory factors involved in mRNA 3 processing, export, translation, and turnover (26), the initiating factor eIF-5A (which may have a role in regulating exonucleolytic decay (27)), and the elongation factor-1 subunit. In contrast, many of the down-regulated proteins in the upf1 strain are involved in DNA replication or RNA transcription. Approximately one-third of the proteins down-regulated in upf1 cells are involved in chromosome and chromatin structure (histones) or transcriptional regulation. These include the Swi3 protein, which is a global activator of transcription required for the induced expression of a large number of genes (28), and Reb1 (29), a sequence-specific DNA-binding protein that recognizes sites within both the enhancer and the promoter regions ...

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...lls are involved in chromosome and chromatin structure (histones) or transcriptional regulation. These include the Swi3 protein, which is a global activator of transcription required for the induced expression of a large number of genes (28), and Reb1 (29), a sequence-specific DNA-binding protein that recognizes sites within both the enhancer and the promoter regions of the rRNA genes as well as sites upstream of many genes transcribed by RNA polymerase II. Also downregulated are the Trm1 and METL (S-adenosyl methionine sythetase) proteins, which are required for methylation of cellular tRNAs (30). While the basis for these observations is unclear, it is possible that the down-regulation of proteins involved in protein translation in xrn1 cells reflects a regulatory circuit responding to the loss of Xrn1p’s role in pre-rRNA processing (31, 32) and that the up-regulation of proteins involved in amino acid biosynthesis in both mutant strains may occur in these strains as a consequence of restoring functional translation of several endogenous nonsense-containing mRNAs encoding enzymes in histidine, arginine, and leucine biosynthesis (33). Relative Changes in mRNA and Protein Levels—He et...

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...f the tag set, but remain isobaric. The example shown is the spectrum obtained from the singly charged [MH] peptide using a 4700 MALDI TOF-TOF analyzer, but the same holds true for any multiply charged peptide analyzed with an ESI-source mass spectrometer. Multiplexed Quantitation Using Isobaric Reagents Molecular & Cellular Proteomics 3.12 1157 at C am bridge U niversity Library on O ctober 16, 2007 w w w .m cponline.org D ow nloaded from tagged peptides. The use of cyclic amines as N-terminal peptide derivatives to simplify the interpretation of MS/MS spectra has been described previously (14,15). We find that the tags behave similarly in both MALDI and ESI, with a tendency to form more abundant and complete b- and y-ion series, while also reducing the proportion of ion current going into less informative fragmentation pathways. Our findings with the six-protein digest mixture show that the derivatization reaction itself is quite straightforward, requiring a simple room temperature reaction of 30 min. Residual reagent is easily quenched by the addition of one or more volumes of water prior to mixing, as decreasing the organic (ethanol) concentration accelerates hydrolysis of residual...

Citation Context

...esses is the identification and measurement of expression levels of relevant sets of proteins. Recently, quantitative approaches utilizing MS and a host of stable isotope-labeling chemistries have emerged (reviewed in Refs. 1 and 2), offering a departure from traditional techniques employing comparative two-dimensional gel electrophoresis. The ICAT quantitative labeling strategy (3, 4) is perhaps the best-characterized method for relative protein quantitation using MS. Other elegant approaches use cell-culture enrichment with a stable isotope-labeled amino acid, including arginine (5), lysine (6), tyrosine (7), and leucine (8), for in vivo incorporation of a mass difference to support relative quantitation. This circumvents potential difficulties surrounding chemical labeling downstream in a comparative experiment. All of these methods impart a mass difference as the basis for quantitation by measurement of relative peak areas of MS and/or MS/MS mass spectra. There are, however, a number of limitations imposed by mass-difference labeling. The mass-difference concept for many practical purposes is limited to a binary (2-plex) set of reagents, and this makes comparison of multiple state...

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...ly increased levels of cellular mRNA (19). One pathway of mRNA decay in yeast involves poly(A) shortening followed by decapping and then 5 to 3 decay. Cells devoid of Xrn1p therefore accumulate mRNAs that have shortened or no poly(A) tails and which may lack the 5 cap structure (20). We find that the xrn1 strain showed specific up-regulation of the ribosome biogenesis protein BMS1 required for the maturation of the 40S ribosomal subunit (21), the SC24 protein required to promote the transport of secretory, membrane, and vacuolar proteins from the endoplasmic reticulum to the Golgi complex (22), and the RNA polymerase II transcription elongation factor TFS2 required for efficient transcription elongation past template-encoded arresting sites. The upf1 strain showed up-regulation of proteins such as the Sec63 protein important for protein assembly in the nucleus and endoplasmic reticulum (23), the Sis1 protein required for normal initiation of translation (24), the polymerase II transcription factor TFIIF that promotes transcriptional elongation (25), and the t-complex protein 1 that acts as a molecular chaperone for protein folding. Proteins Down-regulated in the xrn1 and upf1 St...

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...in required to promote the transport of secretory, membrane, and vacuolar proteins from the endoplasmic reticulum to the Golgi complex (22), and the RNA polymerase II transcription elongation factor TFS2 required for efficient transcription elongation past template-encoded arresting sites. The upf1 strain showed up-regulation of proteins such as the Sec63 protein important for protein assembly in the nucleus and endoplasmic reticulum (23), the Sis1 protein required for normal initiation of translation (24), the polymerase II transcription factor TFIIF that promotes transcriptional elongation (25), and the t-complex protein 1 that acts as a molecular chaperone for protein folding. Proteins Down-regulated in the xrn1 and upf1 Strains— In contrast to the general similarity of proteins up-regulated in the xrn1 and upf1 strains, those proteins that are significantly down-regulated appear to be quite different. Of the 39 proteins down-regulated in the xrn1 strain, only three are common to the upf1 strain. Ontological classification (18) also indicates that the effects of these two mutant strains are substantially different (Table I, Fig. 6). More than half of the proteins down-regulat...

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...addition of 6 volumes of cold acetone (dry ice 20 min). Protein was collected by centrifugation (13,000 g, 5 min), dried in air, and frozen at 80 °C. For digestion, protein was resuspended in digestion buffer (100 mM TEAB, 0.05% w/v SDS) to a final concentration of 1 mg/ml (total protein measured by bicinchonic acid assay (Sigma, St. Louis, MO)). Equal aliquots (500 g) from each lysate were then digested with trypsin overnight at 37 °C (Sigma; 1:40 w/w added at 0 and 2 h) and lyophilized. Labeling with Multiplex Reagents—Synthesis of the four derivatization reagents is discussed elsewhere (11). For each yeast strain, 150 g of total protein was resuspended in 100 l of labeling buffer (0.25 M TEAB, 75% ethanol), after which 1 mg of each isotopically enriched methylpiperazine acetic acid NHS ester was added (1% w/v final) and allowed to react at room temperature for 30 min. Residual reagent was quenched by adding 300 l of water and allowing excess reagent to completely hydrolyze over an additional 30 min, then the three labeled samples were mixed and lyophilized. Cation Exchange Chromatography—The combined peptide mixture was separated by strong cation exchange (SCX) chromatography...

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...16, 2007 w w w .m cponline.org D ow nloaded from (Table I). We compared the current set of differentially expressed proteins with relative expression data from a 2-plex pilot scale study2 of a separate batch of protein lysates from the wild-type and xrn1 strains. This comparison revealed that 86% of up-regulated and 79% of down-regulated proteins (Table I) were identified in the preliminary experiment as up- or down-regulated, respectively. We also compared the current set of differentially expressed proteins to that identified using ICAT for relative protein quantitation in the upf1 strain (16). Because of the substantial difference between workflows, there were proteins unique to each experiment; for example many proteins having two or fewer cysteines (including all major histones) were not observed in the ICAT dataset. However, comparison of the relative expression of proteins common to both experiments proved to be a useful validation of our approach. Of the 85 differentially expressed proteins we observed in the upf1 strain, 49 were also observed by ICAT, and 42 of these were concordant with respect to up- or down-regulation. The average number of peptides identified per protei...

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...eb1 (29), a sequence-specific DNA-binding protein that recognizes sites within both the enhancer and the promoter regions of the rRNA genes as well as sites upstream of many genes transcribed by RNA polymerase II. Also downregulated are the Trm1 and METL (S-adenosyl methionine sythetase) proteins, which are required for methylation of cellular tRNAs (30). While the basis for these observations is unclear, it is possible that the down-regulation of proteins involved in protein translation in xrn1 cells reflects a regulatory circuit responding to the loss of Xrn1p’s role in pre-rRNA processing (31, 32) and that the up-regulation of proteins involved in amino acid biosynthesis in both mutant strains may occur in these strains as a consequence of restoring functional translation of several endogenous nonsense-containing mRNAs encoding enzymes in histidine, arginine, and leucine biosynthesis (33). Relative Changes in mRNA and Protein Levels—He et al. (19) have previously used high-density oligonucleotide microarray analysis to assess global changes in mRNA abundance in the upf1 and xrn1 strains. We have utilized this data to determine whether there is a correlation between the relative chang...