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Molecular & Cellular Proteomics 3:1154-1169, 2004.
© 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Multiplexed Protein Quantitation in Saccharomyces cerevisiae Using Amine-reactive Isobaric Tagging Reagents^*,S

Philip L. Ross, Yulin N. Huang, Jason N. Marchese, Brian Williamson, Kenneth Parker, Stephen Hattan, Nikita Khainovski, Sasi Pillai, Subhakar Dey, Scott Daniels, Subhasish Purkayastha, Peter Juhasz, Stephen Martin, Michael Bartlet-Jones, Feng He^¶, Allan Jacobson^¶ and Darryl J. Pappin^,|,**

From Applied Biosystems, Framingham, MA 01701; Cancer Research UK, London WC2A 3PX, United Kingdom; ^¶ Department of Molecular Genetics and Microbiology, University of Massachusetts, Medical School, Worcester, MA 01655; and ^| Section of Proteomics, Imperial College, Hammersmith Campus, London W12 ONN, United Kingdom

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
We describe here a multiplexed protein quantitation strategy that provides relative and absolute measurements of proteins in complex mixtures. At the core of this methodology is a multiplexed set of isobaric reagents that yield amine-derivatized peptides. The derivatized peptides are indistinguishable in MS, but exhibit intense low-mass MS/MS signature ions that support quantitation. 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 pathways, respectively. We also demonstrate the use of 4-fold multiplexing to enable relative protein measurements simultaneously with determination of absolute levels of a target protein using synthetic isobaric peptide standards. We find that inactivation of Upf1p and Xrn1p causes common as well as unique effects on protein expression.

We have developed a multiplexed set of reagents for quantitative protein analysis that place isobaric mass labels at the N termini and lysine side chains of peptides in a digest mixture. The reagents are differentially isotopically labeled such that all derivatized peptides are isobaric and chromatographically indistinguishable, but yield signature or reporter ions following CID that can be used to identify and quantify individual members of the multiplex set. Absolute quantitation of targeted proteins can also be achieved using synthetic peptides tagged with one of the members of the multiplex reagent set.

In this study, we make use of a 4-fold (4-plex) multiplex strategy to simultaneously determine relative protein levels in three yeast strains and provide a demonstration of the ability to measure the absolute quantity of specific target proteins through the use of internal peptide standards. 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

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
Protein Mixture—
A protein mix consisting of equimolar amounts of BSA, -casein, alcohol dehydrogenase, lysozyme, ß-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 digested with trypsin (1:20 w/w, 50 mM triethylammonium bicarbonate (TEAB), 37 °C, 18 h). Four equal aliquots were treated each with one of the four isotopically enriched methylpiperazine acetic acid N-hydroxy succinimide (NHS) ester reagents by adding 0.5 mg of reagent in ethanol to 50 µl of peptide solution (70% v/v final ethanol) and allowed to react for 30 min at room temperature. The four reactions were combined in various proportions, evaporated to dryness, and quantities representing 500 fmol of each component analyzed by LC-MS/MS as described below.

Yeast Protein Extraction and Labeling—
Log phase cells (75% log; 2.1 x 10⁷ cells/ml) were harvested, frozen, and mechanically lysed by grinding over dry ice. Crude cell lysate was prepared by suspending frozen cellular material (100 mg wet weight) in 1 ml of lysis buffer (0.1 M TEAB, 0.1% v/v Triton X-100, 6 M guanidine), vortexing (1 min), sonicating (5 x 30 s), and pelleting insoluble debris by centrifugation at 13,000 x g for 5 min. Final measured protein concentrations were 3–3.4 mg/ml. Protein was reduced (2 mM TCEP, 37 °C, 1 h), alkylated (5 mM iodoacetamide, 37 °C, 2 h), and precipitated by the addition of 6 volumes of cold acetone (dry ice 20 min). Protein was collected by centrifugation (13,000 x 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 µg) 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 µg of total protein was resuspended in 100 µl 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 µl 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 on an Agilent 1100 HPLC system using a PolySulfoethyl A column (4.6 x 100 mm, 5 µm, 300 Å). Sample was dissolved in 4 ml of SCX loading buffer (25% v/v ACN, 10 mM KH₂PO₄, pH 3, with phosphoric acid) and loaded and washed isocratically for 20 min at 0.5 ml/min to remove excess reagent. Peptides were eluted with a linear gradient of 0–500 mM KCl (25% v/v ACN, 10 mM KH₂PO₄, pH 3) over 15 min at a flow rate of 1 ml/min, with fractions collected at 1-min intervals.

LC-MS Analysis—
Peptide separation was performed on an Ultimate chromatography system (Dionex-LC Packings, Hercules, CA) equipped with a Probot MALDI spotting device. Individual SCX fractions containing 10 µg of protein material were injected and captured onto a 0.3 x 5-mm trap column (3-µm C₁₈ (Dionex-LC Packings, Hercules, CA)) and then eluted onto a 0.1 x 150-mm analytical column (3-µm C₁₈ (Dionex-LC Packings)) using an automated binary gradient (800 nl/min) from 95% buffer A (2% ACN, 0.1% TFA) to 45% buffer B (85% ACN, 5% isopropanol, 0.1% TFA) over 35 min, then 45–90% B in 5 min. For MALDI MS/MS analysis, column effluent was mixed in a 1:2 ratio with MALDI matrix (7 mg/ml--cyano-4-hydroxycinnamic acid) through a 25-nl mixing tee (Upchurch Scientific, Oak Harbor, WA) and spotted in 16 x 16 spot arrays. MALDI plates were analyzed on an ABI 4700 (Applied Biosystems, Framingham, MA) proteomics analyzer. Peptide CID was performed at a collision energy of 1 kV and a collision gas pressure of 1.5 x 10^–6 Torr. For electrospray analysis, an Ultimate LC system interfaced to a Qstar Pulsar (Applied Biosystems-MDS Sciex) mass spectrometer was used. The LC conditions were similar to those used for LC-MALDI, with peptides separated at a flow rate of 300 nl/min over a 75-µm x 150-mm C₁₈ column (Pepmap; Dionex) using a 2-h gradient of 5–35% B (A, 2% ACN/0.1% formic acid; B, 98% ACN/0.1% formic acid). Survey scans were acquired from m/z 300–1,500 with up to three precursors selected for MS/MS from m/z 90–2,000 using dynamic exclusion. A rolling collision 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). S-acetamido, 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 (N- and 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 was then housed in an Access (Microsoft, Redmond, WA) database for grouping of results into proteins and calculation of ratios and standard deviation. Abundance ratio calculations included corrections for overlapping isotopic contributions (both natural and enriched ¹³C components).

	RESULTS AND DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
Features of Multiplexed Tagging Chemistry—
The components of the multiplexed derivatization chemistry are introduced in Figs. 1 and 2. In preliminary studies, we used a reduced and alkylated protein digest mixture as a simple model system to validate the labeling protocol and the usage of MS/MS signature ions for quantitation. A digest mixture of six proteins was split into four identical aliquots. Each was then labeled with one of the four isotopically labeled tags, and the derivatized digests combined in mixtures of varying proportions. The multiplex isobaric tags produce abundant MS/MS signature ions at m/z 114.1, 115.1, 116.1, and 117.1, and the relative areas of these peaks correspond with the proportions of the labeled peptides. We have found that this mass range also has minimal contamination with background low-mass fragments produced from CID fragmentation of peptides using either MALDI or ESI-based tandem mass spectrometers.

View larger version (29K): [in this window] [in a new window]   FIG. 1. A, diagram showing the components of the multiplexed isobaric tagging chemistry. The complete molecule consists of a reporter group (based on N-methylpiperazine), a mass balance group (carbonyl), and a peptide-reactive group (NHS ester). The overall mass of reporter and balance components of the molecule are kept constant using differential isotopic enrichment with 13C, 15N, and 18O atoms (B), thus avoiding problems with chromatographic separation seen with enrichment involving deuterium substitution. The number and position of enriched centers in the ring has no effect on chromatographic or MS behavior. The reporter group ranges in mass from m/z 114.1 to 117.1, while the balance group ranges in mass from 28 to 31 Da, such that the combined mass remains constant (145.1 Da) for each of the four reagents. B, when reacted with a peptide, the tag forms an amide linkage to any peptide amine (N-terminal or amino group of lysine). These amide linkages fragment in a similar fashion to backbone peptide bonds when subjected to CID. Following fragmentation of the tag amide bond, however, the balance (carbonyl) moiety is lost (neutral loss), while charge is retained by the reporter group fragment. The numbers in parentheses indicate the number of enriched centers in each section of the molecule. C, illustration of the isotopic tagging used to arrive at four isobaric combinations with four different reporter group masses. A mixture of four identical peptides each labeled with one member of the multiplex set appears as a single, unresolved precursor ion in MS (identical m/z). Following CID, the four reporter group ions appear as distinct masses (114–117 Da). All other sequence-informative fragment ions (b-, y-, etc.) remain isobaric, and their individual ion current signals (signal intensities) are additive. This remains the case even for those tryptic peptides that are labeled at both the N terminus and lysine side chains, and those peptides containing internal lysine residues due to incomplete cleavage with trypsin. The relative concentration of the peptides is thus deduced from the relative intensities of the corresponding reporter ions. In contrast to ICAT and similar mass-difference labeling strategies, quantitation is thus performed at the MS/MS stage rather than in MS.

View larger version (26K): [in this window] [in a new window]   FIG. 2. Example MS/MS spectrum of peptide TPHPALTEAK from a protein digest mixture prepared by labeling four separate digests with each of the four isobaric reagents and combining the reaction mixtures in a 1:1:1:1 ratio. Components of the spectrum illustrated are (i) isotopic distribution of the precursor ([M+H]+, m/z 1352.84), (ii) low mass region showing the signature ions used for quantitation, (iii) isotopic distribution of the b6 fragment, and (iv) isotopic distribution of the y7 fragment ion. The peptide is labeled by isobaric tags at both the N terminus and C-terminal lysine side chain. The precursor ion and all the internal fragment ions (e.g. type b- and y-) therefore contain all four members of the tag set, but remain isobaric. The example shown is the spectrum obtained from the singly charged [M+H]+ 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.

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 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 reagent. Hydrolysis is essentially complete within an additional 30 min at room temperature. Comparison of protein digest mixtures of known proportions (Fig. 3) gave accurate ratios (<6% error) and standard deviations less than 23% across two orders of magnitude. All observed peptides were confirmed as fully derivatized at N termini and lysine side chains.

View larger version (31K): [in this window] [in a new window]   FIG. 3. Summary of relative protein measurements for (i) 1:1:1:1 and (ii) 1:5:2:10 mixtures of a six-protein digest. Ratios were expressed relative to the peak area at 117.1. Insets show examples of the signature ion regions from peptides mixed 1:1:1:1 and 1:5:2:10. Intra-protein averages are compiled from the top-ranked significant peptides labeled at N termini and lysine side chains (Mascot score > 95% confidence, rank = 1).

View larger version (19K): [in this window] [in a new window]   FIG. 4. Depiction of overall workflow used for multiplexed comparative analysis of the wild-type, upf1, and xrn1 strains. Equivalent amounts of whole-cell lysates were treated in parallel workflows employing standard protocols for reduction and alkylation, trypsin digestion, and derivatization (see "Materials and Methods"). A number of internal standards were derivatized with a fourth reagent, and the four samples were mixed, combined with cation exchange loading buffer and subjected to SCX chromatography followed by capillary reverse-phase HPLC coupled to MALDI-MS/MS or ESI-MS/MS.

Protein Identification—
Proteins were identified on the basis of having at least one peptide whose individual ion score was above the 95% confidence threshold (p < 0.05) and also identified as the top-ranked matching sequence for that spectrum (12). We further restricted the number of identifications made by imposing spectrum and peptide-level nonredundancy such that any given peptide and any given MS/MS spectrum was linked to only one protein. Using these criteria, 1,217 unique proteins were identified from 4,500 peptides. More importantly, 685 of these proteins were identified by two or more of these significant peptides. Further statistical analysis for determining up- or down-regulation of protein expression levels was performed only on this latter group of 685 proteins (i.e. protein identifications made on single, unique peptides were discarded).

Protein Quantitation—
The individual ratios of identified peptides from the upf1 and xrn1 strains compared with the wild-type strain were computed from signature ion peak areas using the formula: area(mutant)/(area(mutant) + area(wild type)). All protein expression values thus fall between 0 and 1, with a "no-change" 1:1 ratio = 0.5. The global all-peptide average and standard deviations were 0.503 (±0.084) and 0.485 (±0.044) for the xrn1 and upf1 strains, respectively. These values indicate a high degree of consistency between these three strains, suggesting that the parallel, peptide-based workflow did not introduce significant variability into the measurements. Peptide ratios were also grouped into proteins and averaged to arrive at protein-level ratios for those 685 proteins having two or more significant scoring peptides. The average of the protein-level standard deviations was 0.055 and 0.034 for the xrn1 and upf1 strains, respectively. In other words, there was a high degree of concordance between individual peptides contributing to the relative quantitation of any given protein.

From the global mean and standard deviation for each strain we identified proteins whose average expression ratios fell outside of ±1 standard deviation from the global mean. We further excluded proteins whose individual standard deviation (i.e. between peptides) was greater than 0.1. From this, a list of up- and down-regulated proteins was generated for the xrn1 and upf1 strains (Table I). Using these criteria, 62 and 48 proteins were considered up-regulated and 23 and 39 proteins down-regulated in upf1 and xrn1, respectively (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 protein also increased from 2 peptides/protein in the ICAT study to 4.5 peptides/protein in the current work.

View this table: [in this window] [in a new window]   TABLE I Summary of significant protein expression changes in multiplex Xrn1-Upf1-WT comparative study

The experiment also demonstrated absolute quantitation of selected proteins (Fig. 5A). In this example, 1 pmol of a synthetic tryptic peptide (ILESHDVIVPPEVR from carbamoyl phosphate synthetase) was labeled with the 117-reporter isobaric reagent and combined with the yeast strains prior to cation exchange chromatography. The peptide was identified 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 µg 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), where the absence of a signature ion at m/z 114.1 established the loss of this protein.

View larger version (52K): [in this window] [in a new window]   FIG. 5. Example signature ion regions from four MS/MS spectra illustrating the degree of consistency in relative quantitative measurement of (A) an up-regulated protein (carbamoyl phosphate synthetase). Illustrated are the signature ion regions of four identified tryptic peptides (from a total of 11) that show consistency of measurement between the three strains. For all 11 peptides identified, the fold increase of this protein in the xrn1 strain was 2.15 relative to wild type, with an S.D. of <12%. This protein was also used to demonstrate an absolute quantitative measurement using an internal spiked synthetic tryptic peptide (ILESHDVIVPPEVR) labeled with the 117-reporter isobaric reagent. B, highlighted signature ion region showing confirmation of deletion of the Xrn1p protein in the xrn1 yeast strain by quantitative measurement of the identified tryptic peptide IGPMEAIATVFPVTGLVR (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.

Proteins Up-regulated in the upf1 and xrn1 Strains—

xrn1

upf1

xrn1

xrn1

upf1

upf1

upf1

xrn1

View larger version (43K): [in this window] [in a new window]   FIG. 6. Display of ontology profile from an ontology search (18) of significant up- and down-regulated proteins observed in the multiplex yeast experiment. Protein classification groups were identified as significant by comparison with the remaining identified proteins that were not significantly up- or down-regulated (p < 0.05). This approach determines whether the chosen up- or down-regulated proteins are distributed randomly with respect to the classification of the remaining (600) identified proteins whose expression levels do not change.

xrn1

upf1

xrn1

upf1

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-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 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 of the rRNA genes as well as sites upstream of many genes transcribed by RNA polymerase II. Also down-regulated 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 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 relative changes in protein levels determined in our analyses of the wild-type, upf1, and xrn1 extracts. Fig. 7A shows that, for those proteins showing significant 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.

View larger version (63K): [in this window] [in a new window]   FIG. 7. Changes in mRNA levels do not correlate with changes in protein levels in upf1 and xrn1 cells. Scatter plots were used to compare the average mRNA and protein expression ratios in upf1 (A) and xrn1 (B) cells. Expression ratios are defined as the fold-change in mutant versus wild-type cells. Average mRNA expression ratios are from He et al. (19). Protein expression ratios are derived from the upf1 data (85 proteins) and xrn1 data (87 proteins) listed in Table I. Pair-wise comparisons are plotted on a logarithmic scale, with the middle line indicating the line of equivalence and the outer two lines indicating 2-fold differences in expression.

	CONCLUSIONS

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

In this study, we used the reagents to compare global protein expression in wild-type yeast and the isogenic upf1 and xrn1 mutant strains that are defective in the nonsense-mediated mRNA decay and the 5' to 3' decay pathway. We find that inactivation of Upf1p and Xrn1p cause both common as well as distinct effects on protein expression. Both mutant strains show increased expression of a common set of proteins involved in amino acid biosynthesis and general nitrogen metabolism. The upf1 strain showed specific down-regulation of proteins involved in DNA replication and RNA transcription, whereas the xrn1 strain exhibited specific down-regulation of components of the translation apparatus, including ribosomal proteins and translation factors. Comparison between mRNA changes and protein changes of these yeast strains showed no significant correlation.

The isobaric nature of the tags permitted the simultaneous comparison of multiple yeast strains and added synthetic peptide internal standards in a single two-dimensional-LC-MS experiment, with no increase in chromatographic or MS complexity. Importantly, ratio measurements for all the identified peptides was 100% for all strains. Measured expression ratios demonstrated high consistency, and intra-protein peptide mean and standard deviations were highly reproducible (15–17%). The mixed, multiplex nature of the experiment removes any quantitative variability from chromatography that may be seen in sequential two-dimensional LC-MS analyses of individual peptide mixtures (36), and peptide coverage is significantly increased relative to ICAT. The tagging chemistry is global in that any peptide with a free amine can be labeled and measured. This should enable strategies that seek to isolate and quantify specific classes of peptides (e.g. phosphopeptides) that were essentially impossible using the ICAT cysteine-selective chemistry.

	ACKNOWLEDGMENTS

 
We gratefully acknowledge the contributions of Ralph Casale, Ivar Jensen, and Jim Coull for help and advice. We would also like to thank Dan Knapp for his efforts in deciphering the strategy used for isotope coding.

	FOOTNOTES

 
Received, September 16, 2004, and in revised form, September 22, 2004.

Published, MCP Papers in Press, September 22, 2004, DOI 10.1074/mcp.M400129-MCP200

¹ The abbreviations used are: PTM, post-translational modification; TCEP, Tris-(2-carboxyethyl)phosphine; TEAB, triethylammonium bicarbonate; NHS, N-hydroxy succinimide; SCX, strong cation exchange.

² Pilot studies were conducted as a 2-plex experiment using 100 µg of total protein with similar conditions for protein extraction, digestion, labeling, chromatography, and MS.

^* Part of this work was supported by National Institutes of Health Grant GM27757 (to A. J.).

^S The on-line version of this manuscript (available at http://www.mcponline.org) contains supplemental material.

^** To whom correspondence should be addressed: Applied Biosystems, 500 Old Connecticut Path, Framingham, MA 01701. Tel.: 508-383-7484; Fax: 508-383-7813; E-mail: pappindj{at}appliedbiosystems.com

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TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 

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