Mass spectrometry analysis of proteome-wide proteolytic post-translational degradation of proteins

doi:10.1021/ac800077w

Anal Chem. Author manuscript; available in PMC 2009 August 1.

Published in final edited form as:

Anal Chem. 2008 August 1; 80(15): 5819–5828.

Published online 2008 June 26. doi: 10.1021/ac800077w

PMCID: PMC2716136

NIHMSID: NIHMS113148

Mass spectrometry analysis of proteome-wide proteolytic post-translational degradation of proteins

Yufeng Shen,¹ Kim K. Hixson,² Nikola Tolić,² David G. Camp,¹ Samuel O. Purvine,² Ronald J. Moore,¹ and Richard D. Smith^1,²

¹Biological Science Division, Pacific Northwest National Laboratory, Richland, WA 99352

²Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA 99352

Requests for materials should be addressed to Yufeng Shen (Email: yufeng.shen/at/pnl.gov) or Richard D. Smith (Email: dick.smith/at/pnl.gov)

The publisher's final edited version of this article is available at Anal Chem

See other articles in PMC that cite the published article.

Abstract

Protein proteolytic degradation is an essential component to proper cell function and its life cycle. Here, we study the protein degradation in yeast Saccharomyces cerevisiae cells on a proteome-wide scale by detection of the intermediate peptides produced from the intracellular degradation of proteins using sequencing-based tandem mass spectrometry. By tracing the detected ~1,100 peptides and their ~200 protein substrate origins we obtain evidence for new insights into the proteome-wide protein selective degradation in yeast cells. This evidences shows that the yeast cytoplasm is the largest pool for the degradation of proteins with both biochemical and geometric specificities, while the yeast nucleus seems to be a proteolysis-inert organelle under the condition studied. Yeast V-ATPase subunits appear to be degraded during their disassembly, and yeast mitochondrial proteins functioning as precursors, transport carriers and gates are preferentially degraded. Ubiquitylation may be unnecessary for the proteasomal degradation of yeast cytoplasmic regulatory and enzyme proteins according to our observations. This study shows that the intracellular peptides are informational targets for directly probing the protein degradation-involved molecular mechanisms and cell biology processes.

Protein degradation is a post-translational modification necessary for protein quality control and cell survival. In eukaryotic cells degradation of proteins is primarily carried out in either the proteasomes or lysosomes. Proteasomes,¹^,² are responsible for protein quality control by breaking down misfolded proteins,³ and they have a well-defined geometric proteolysis-catalytic center⁴^,⁵ that breaks down the proteins to produce peptides with specific lengths.⁶^,⁷ Ubiquitylation⁷^,⁸ of proteins is the typical initial step for protein degradation through the proteasomes (note: ubiquitylation also functions for non-proteolytic purposes¹⁰). Resultant peptides from proteasomal degradation can then be further cleaved into amino acids by cytosolic aminopeptidases.¹¹ Lysosomes, on the other hand, are membrane bound organelle-like structures containing multiple proteases that degrade protein aggregates and membrane proteins.³^,¹² Proteins to-be-degraded are transported to the lysosome through e.g., the ATGs/signaling/regulating systems¹³^,¹⁴ and the following proteolyses are catalyzed by endoproteinases¹⁵^,¹⁶ that are matured and activated within the lysosome. The resultant peptides continue to break down into amino acids by the lysosomal aminopeptidases and carboxypeptidases,¹⁶^,¹⁷ and the products (e.g., amino acids and others such as sugars, cholesterols) are then transported back out into the cytosol by the solute transporters in the lysosomal membrane and can then be recycled as nutrients making this degradation process important for cell survival when cells are subjected to stresses such as nutrient limitation.¹³^,¹⁴

Protein degradation has been primarily studied using the methods based on radioactivity,³ which have proven to be informative on an overall level, but provided little information regarding the degradation steps occurring to individual proteins. Such information currently has been gained by performing in vitro experiments⁶^,⁷^,¹³^–¹⁷ with individual model proteins/peptides and proteases, but evidences provided from these in vitro experiments do not represent the true in vivo scenarios due to the inability of a few single proteins to truly mimic the regulatory processes and complexity of degradation occurring to multiple proteins at the same time within a cell. Additionally, current ideas on protein degradation concede that in vivo monitoring of overall cellular protein degradation is less possible because it is believed that intermediate peptides being produced by the proteasomes or lysosomes are broken down so quickly into amino acids by peptidases that the sequence of protein degradation is undetectable except for special cases such as with the major histocompatibility complexes.¹⁸^,¹⁹ The proteolytic degradation of proteins in cells on the proteome-wide level basically is still not understood fully.

In this work, we introduce a mass spectrometry (MS)-based method to study proteome-wide protein degradation. Yeast Saccharomyces cerevisiae was used as a model organism due to its simplicity and mechanistic homology to higher eukaryotic cells. Various aspects of information for yeast are now available including protein sequences (ftp://genome-ftp.stanford.edu/pub/yeast/data_download/sequence/GenBank/), localization,²⁰ expression levels,²¹ life times,²² and proteolytic activities of proteasomal and lysosomal (vacuolar) proteinases and peptidases (summarized in Supplementary notes). We demonstrate that protein degradation can be probed on a proteome-wide scale by sequencing the protein degradation intermediate peptides with high-precision tandem MS (i.e., MS/MS). The obtained information provides evidence for insights and better understanding of the molecular mechanisms and processes involved in protein proteolytic degradation.

RESULTS

Identification of intracellular peptides from yeast lysate using sequencing-based mass spectrometry

Figure 1 shows a LC-MS display of molecular species having masses <10,000 u from a yeast cell lysate. In this display, ~8,000 different molecular species exist. Identification of these displayed molecules was accomplished with accurate MS/MS data that were concurrently acquired during the LC-MS measurements (see Methods). Differing from the conventional probability-based scoring approaches, we developed a sequencing-based UStag method (see Methods) to achieve unambiguous identification of various lengths and termini of peptides with expected/unexpected post-translational modifications. In total, we identified 1,111 peptides (Supplementary Table 1) having lengths of 6–100 amino acids (those having ~20 amino acids were most frequently observed, see Supplementary Figure 1). Among these peptides, 59 were found as modified with mutations, acetylations, amidations, etc. The correctness of the identifications was evidenced with spectral similarity, correct molecular masses and isotopic envelope patterns, correct fragmental masses, correct residues, and the database uniqueness of the sequences constructed from the consecutive fragments and the residues measured even with consideration of various possible modifications (see Methods). The random false positives were tested by searching all acquired MS/MS spectra (30,760 in total) against a reverse ordered yeast protein sequence database using the same method as that used for peptide identification, and no false positives were found except for one with a symmetric sequence (i.e. EKAKE, from YAL038W).

Figure 1

LC-MS display of molecular species having molecular masses of 400–10,000 u in a yeast Saccharomyces cerevisiae lysate. Species with >3 MS scans were counted. The mass accuracies for both amino acid sequencing and individual mass peaks (more ...)

Peptide origins found from the yeast cell lysate

Each peptide identified was examined to determine its possible biochemical/biological origins. These origins include a protein maturation process (protein cleavage that transforms inactive proteins into active conformations), a partially assembled protein (a protein in the middle of being synthesized by a ribosome at the time of cell harvest), a product of a protein localization process, a part of a protein signaling process, or a product of a protein degradation process. The identified peptides that were a component of the first 70 amino acids of the full predicted sequence of each protein were analyzed using SignalP²³ to examine the signal peptides, and only 1 signal peptide was found from the yeast protoplast secreted protein 2 precursor. The possible products from proprotein convertases (Kex2 in yeast),²⁴ which cleaves polypeptide sequences at dibasic amino acid sites (i.e., RR, RK, KR, and KK) to convert proproteins into mature protein forms and which leaves propeptides behind, were examined; and 4 of the identified peptides were potential candidates due to the dibasic characteristic cleavage sites. However, these 4 peptides were more likely the products of degradation or partially synthesized sequences, as they were found also to be the components of peptide ladders which were typically seen from protein degradation shown in this study. We did not observe any evidence of intramembrane proteolysis under the growth condition studied, and it is unknown whether or not yeast Saccharomyces cerevisiae can generate <10,000-u protein fragments from protease cascade processing for these membrane proteins. The protein synthesis, starting from the protein N-termini, may also generate N-terminal peptides due to the interruption of synthesis at the time of cell harvest, and we found that peptides corresponding to 15 proteins were potential products of the protein partial synthesis as they were not accompanied with other peptide ladders.

Figure 2 is an example of the peptide ladder pattern observed for a yeast protein (phosphoglycerate kinase) in the cell lysate. Observed is a sequential cutting of the protein into smaller and smaller pieces from the long peptides (the longest peptides detected having 70 amino acids for this example). The peptides were verified to be present in the lysate before LC-MS analysis as these ladder sequences eluted at different LC elution time profiles (not shown). Non-degradation related intracellular proteolysis processes (e.g., N-terminal signal peptide, propeptides, and partial protein synthesis etc.) do not explain these ladders as many specific cleavages were located within the middle of the sequence or near the C-terminus of the protein. These ladders were similar to the proteasome peptide map observed in previous in vitro experiments with model proteins,²⁵ and the peptides detected from our experiment have a large range in size (e.g., up to 75-AA residues for the shown example) and display cleavage preferences (e.g., various peptides in the ladders starting at specific points).

Figure 2

Peptide ladders showing yeast phosphoglycerate kinase sequences occurring in the cells at the time of cell harvest. The peptides observed had lengths of 12–70 amino acid residues; the curved sequence unnecessarily represents the protein real folding. (more ...)

Organellar selectivity of the detected peptides’ protein origins

We examined the protein origins for the peptides identified and found that 205 yeast proteins were the source of the 1,111 peptides identified (Supplementary Table 1). These proteins have expressions ranging from low (e.g., ~150 molecules/cell for Hsp 10 kDa) to high (e.g., ~1 × 10⁶ molecules/cell for fructose-bisphosphate aldolase) levels²¹ and half-lives ranging from short (e.g., 10 min for vacuolar ATPase subunit E) to long (e.g., >1,500 min for viral protein U-binding protein) ones.²² The number of peptides identified did not necessarily correlate with the protein expression levels; e.g., only 2 peptides were detected for both Hsp SSB2 (1 × 10⁵ molecules/cell documented) and the 60S ribosomal protein L3 (450 molecules/cell documented).

We next looked for the sub-cellular location of these proteins using information from http://db.yeastgenome.org/cgi-bin/search/featureSearch. Of the 205 proteins found, 87 co-localize in multiple organelles and 118 belong to a single organelle (Supplementary Table 2). Figure 3 shows the organellar distribution of the 118 single organelle-specific proteins. It clearly shows that these single organelle-specific proteins showed a high-selectively from a specific yeast organelle such as cytoplasm and mitochondria, while other organelles including the yeast nucleus, cell wall, and cytoplasmic membrane did not release any single peptide (either hydrophilic or hydrophobic one). This finding excludes the possible contribution of artifact proteolysis (see Discussion section below) during and after the cell lysis process, which was completed in ~200 sec in the presence of protease inhibitors (see Method), and suggests that the identified peptides were products from the breakdown of specific proteins in specific locations. Excluding those from potential non-proteolysis processes described above, we ascribe the identified peptides to the products of the protein intracellular in vivo degradation. Additionally, the organelle selectivity observed for protein breakdown also excluded lysosome-involved proteolyses as these types of proteolyses are preferential in the breakdown of membrane proteins,³^,¹³^,¹⁴ which were not found. This claim is consistent with fact that there was a lack of inducers to start the lysosome-involved apoptosis and autophagy from the nutrient-rich, rapid growth environments that the yeast cells were exposed to at the time of harvest in this study.

Figure 3

Localization of 118 yeast proteins observed with the existence of their intracellular peptides. Proteins observed in each organelle are described in the following sections.

Proteolytic degradation of yeast cytoplasmic proteins

Yeast cytoplasm was found to contain the largest pool where the breakdown products of 77 of 118 single organelle-specific proteins were found. Ribosomal, stress-response, regulatory, and enzymatic proteins contributed to the large portion of peptides found from cytoplasmic proteins (Table 1). Most ribosomal and stress-response protein products found were ubiquitylation proteins (http://ubiprot.org.ru/), and their proteolysis may be explained by the ubiquitylation-proteasome pathway. In contrast, most proteins annotated as regulatory and enzymatic in function were not ubiquitylation proteins. Proteasomes are the major proteolytic machine in yeast cytoplasm, which leads us to hypothesize that the degradation of these regulatory and enzymatic proteins found (or some of those) should be carried out through proteasomes without ubiquitylation. Next we examined the proteolytic specificity of the peptides detected (Figure 4). At the amino acid cleavage site P1, trypsin-like and chymotrypsin-like proteolyses, i.e., the activities of yeast proteasome β2/PUP1 and β5/PRE2, were observed. The cleavage preferences for Leu and Ala were also observed, which agrees with the specificity of yeast proteasomal β1/PRE3, β2/PUP1, and β5/PRE2.²⁵ Removal of Met from the protein N-terminus²⁶ was responsible for the high frequency observation of P1 Met (of the 57 P1 Met found, 47 were located at the N-termini). At P1′ we found the cleavage preference was for amino acids having short side chains (e.g. Gly, Ala, Ser, and Thr), i.e., sites with small steric hindrance. This steric driven preference is likely imposed by proteasomes where a partially folded protein “loop”²⁷ at the location of the small amino acid enters the proteasome more easily and these protein portions thus can access the tiny catalytic cavity²⁸ that allows for the protein hydrolysis. The protein “loop” model can also elucidate our observations of peptides significantly longer than those predicted by the “molecular ruler” (i.e., generation of ~8-residue peptides) theory of proteasomal degradation⁶^,⁷ with additional consideration of kinetic factors²⁹ for proteolysis of the cellular proteins.

Table 1

Properties of the yeast cytoplasm-specific proteins observed from intermediate peptides detected in this study.^*

Figure 4

Proteolytic cleavage preferences of yeast Saccharomyces cerevisiae cytoplasmic proteins. 462 peptides observed for the 77 cytoplasm-specific proteins were analyzed. The cleavage sites were defined as follows: NH2- … –P1 ↓ P1′– (more ...)

The yeast nucleus is not the active site for protein proteolytic degradation

In contrast to observations of a significant number of degraded cytoplasm-specific proteins, no single peptide from the breakdown of any nucleus-specific protein was found. Compared to ~1600 cytoplasm-specific proteins, yeast Saccharomyces cerevisiae is predicted to have ~900 nucleus-specific proteins that have a variety of documented lifetimes (e.g., 2–>13,000 min) and expression levels (e.g., 100–>10,000 molecules/cell). The absence here in observation of nucleus-specific protein degradation products suggests that the yeast nucleus itself is a proteolysis-inert site in the cells studied. The 26S proteasomes seem not to be located in the yeast nucleus (http://www.nature.com/nrm/poster/ubiquitin/poster.pdf), while 20S proteasomal subunit complexes have been observed to be imported into the yeast nucleus.³⁰ Our results here reveal that the 20S complexes imported (if it happened) were not matured or functional in the nucleus to warrant proteolysis.

Nuclear proteins of yeast that co-localized in other proteolysis-active organelles or locations were observed to be degraded in this study. For example, 55 of the 58 yeast nucleus-related proteins (as defined by yeastgenome.org, see Supplementary Table 1) were co-localized within the highly active cytoplasm; the other 3 nucleus-related proteins found included 2 short-lived nucleus pore proteins NSP1³¹ (co-localized with mitochondrion) and NUP2³² (co-localized with the endoplasmic reticulum and cytoskeleton) and a yeast protein transporter histone (co-localized within the vacuole). Proteolytic degradation of these nucleus-related yeast proteins are thus hypothesized to take place by the proteolysis-active enzymes found in the cytoplasm (containing 26S proteasomes) or other organelles.

Yeast V-ATPase degradation

V-ATPase is composed of a membrane associated-V₀ domain and a peripheral membrane protein complex that makes up the V₁ domain.³³ Yeast V-ATPase V₀ domain contains 6 subunits (a, d, c, c’, c” and e) and the V₁ domain is an assembly of 8 subunits A–H. We detected intermediate peptides from the breakdown of all 7 V₁ subunits including A, B, C, E, F, G, and H (H was identified only from its N-terminal peptides, see Supplementary Table 1), only missing the stalk subunit D that connects 7 other subunits to the V₀ domain³³ although the subunit D protein has a higher expression level (8,500 molecules/cell) than some other subunit proteins (4,000 and 2,300 molecules/cell for F and G, respectively).²¹ (The lifespan of subunit D is not available, while the other 7 subunit proteins have half-lives of 10–300 min.²²) Peptide products from the V₀ membrane domain subunit proteins were not found. From these observations, we deduce that degradation of the V₁ subunit proteins is a process where subunits A−C and E−H first uncouple (dissemble) from the D, become a free floating state in cytoplasm, and then are degraded there by cytoplasmic proteasomes (Figure 5). Subunit D remains connected to the V₀ domain while the rest of the V₁ subunits disassemble, which protects subunit D from the degradation. It is unlikely that the degradation of the V-ATPase subunit proteins happened during their transport, localization, and assembly processes; otherwise all subunits including D should be equally degraded and detectable. The observed degradation of the V₁ subunit proteins also reveals that the assembly-disassembly of the V-ATPase may actually be irreversible and require new protein synthesis to make up a deficiency of the degraded subunit proteins which maintain normal function of the vacuolar proton pump. In other words, it appears as though the subunits are not simply re-used from disassembled subunits to reform the complex and make it active again.³⁴

Figure 5

Observed degradation of the yeast V-ATPase subunits suggesting that the process happens after disassembly of subunits from subunit D. The V-ATPase structure diagram was redrawn based on ref. (³³).

Selectivity/specificity for proteolysis of yeast mitochondrial proteins

Yeast mitochondria with their own proteolytic activities³⁵ are the second largest pool for protein degradation observed in this study. In total, we found 28 mitochondrion-specific proteins (Supplementary Table 1) with the detection of their degradation-produced peptides, and Figure 6 shows the compartmental locations of these proteins. Of the 28 proteins, 17 were located in the mitochondrial matrix (MM), proteolytic degradation of which should be catalyzed by proteasome-like ClpP/ClpX and Yta12p.³⁵ The transport carrier and gate proteins in the mitochondrial inner membrane (MIM, containing proteolytic protease Yta12p³⁵) were selectively degraded, including 4 transport-related proteins, 2 ADP/ATP carrier proteins, 2 CcO proteins, 1 acryl carrier protein, 1 TIM 23 subunit protein (TIM 23 was found with only N-terminal peptide ladders), and the tricalbin-3 (MIM-attached, facing the MM side). For the mitochondrial outer membrane (MOM) compartment, only transport gate proteins (OM45 and TOM70³⁶^,³⁷) appeared to be degraded. Degradation of the mitochondrial intermembrane space (MIMS) proteins was not found, which agrees with the lack in observation of the proteolytic proteases in yeast MIMS.³⁵ The yeast mitochondrial proteases’ preference is determined to be in the following order: Lys > Leu > Ser > Phe > Arg = Ala > Val > Gln > … at P1 and Gly>Arg>Ser>Lys>Glu>Thr=Ala>Asp>… at P1′ according to the peptides detected.

Figure 6

Distribution of the degraded yeast mitochondrial proteins. The translocation diagram was modified based on ref. (³⁵).

Proteolytic degradation of other organellar proteins observed

In addition to the major proteolytic activities observed above, proteolytic degradation of a few cytoskeleton- and endoplasmic reticulum (ER)-specific proteins was also found. The 3 cytoskeleton-specific proteins found included actin, actin-capping protein (CAP1), and protein cofilin promoting actin filament remodeling and turnover,³⁸^,³⁹ all of which play key roles in endocytosis. The 3 ER-specific proteins found include the secretory-regulating protein BFR1, regulator protein GRP 78 (a member of the Hsp70 family) involved in incorrectly folded protein regulation,⁴⁰^,⁴¹ and a sterol-acyltransferase 2.

DISCUSSION

Using yeast Saccharomyces cerevisiae as a model organism, we demonstrated that thousands of molecular species with masses of <10,000 u were detectable for cells at the time of harvest (shown in Figure 1). Identification of peptides from this mixture challenges the method for identification accuracy without bias against the peptide’s terminus and size. The accurate MS/MS sequencing-based unique sequence tags method used in this study, yielding extremely few random false positives, resolves this challenge. This sequencing-based method is desirable to probe the protein proteolysis processes and the activities and specificity of the related proteolytic proteases on a proteome-wide scale (as demonstrated in the Results section), which also covers the peptide scope of peptodomics⁴³ (study of intercellular peptides) and degradomics⁴⁴ (study of enzyme activity). Using the reliable identification method, we identified ~1,100 peptides among the relatively small molecules observed from the cell lysate. These peptides have a range of sequence lengths from 6 to 100 amino acid residues and show up with specific cleavage sites and show up overall as an array of protein pieces that produce sequence ladders (see Figure 2).

Both in vivo protein proteolysis (i.e., intracellular degradation) and protein breakdown during/after the breaking of the cells and the releasing of proteins and their degradation products from their specific cellular locations (i.e., extracellular proteolysis or artifacts) can contribute peptides in the lysate of cells. In this study, we used pressure cycling technique (PCT⁴⁵) for the cell lysis and peptide/protein extraction. There was no evidence that the mechanical processes applied to the cells (including the cell freeze-thaw and pressurized lysis) attributed to any sort of protein cleavage, and furthermore, the cleavage selectivity pattern seen in this study (Figure 4B). (In fact, the high pressure has been shown to only alter protein higher structures, e.g., protein folding/unfolding and stability of protein aggregates.⁴⁶) The extracellular proteolysis of proteins during sample processing are expected to favor detection of high-abundance proteins located in various locations, but the proteins assigned from the peptides detected in this study had little correlation with the proteins’ abundances (Supplementary Figure 2). For example, the highly-abundant proteins located in the yeast nucleus, e.g., yeast histone H4 (GNL030W) with a documented abundance of >600,000 molecules/cell and a GRAVY value of −0.5, were not observed. The lack in detecting such high-abundance proteins is less possible from the incomplete disruption of cells by PCT, as PCT has been demonstrated as being able to release proteins that are difficult to be released by traditional bead beating, pulverization under liquid nitrogen, and sonication methods widely used for sample preparation in 2D gel.⁴⁵ Additionally, many highly-abundant cytoplasmic proteins (e.g., >350 proteins having abundances of >10,000 molecules/cell), including the most abundant cytoplasmic protein CWP2 (YKL096W-a, ~1,600,000 molecules/cell), were not in our protein identification list (Supplementary Table 1) where the abundance of proteins identified can be as low as ~450 molecules/cell. This evidence reveals that post-lysis artifact proteolysis by the released proteases during the step-by-step PCT cell breaking and protein lysis contributed little to the peptides detected, and the proteolysis that occurred within the cells (or intracellular degradation of proteins) at the time of cell harvest should be responsible for the peptide observations.

Through detection of intracellular peptides, we now can “see” the proteome-wide protein post-translational proteolytic degradation. It was observed that only proteins located in some special organelles and compartments contributed the peptides that were detected (see Figure 3, Figure 5, and Figure 6). The potential influence of use of a hydrophilic PCT solvent for extraction of peptides analyzed (see Methods section) on such observations were investigated by examination of the hydrophobicity of peptides detected. The GRAVY values for the peptides detected were distributed over a range from −1.9 to 1.7 (Supplementary Figure 3). Both soluble and insoluble (membrane) proteins can generate hydrophilic and moderately hydrophobic peptides located in this GRAVY range.⁴⁷ The peptides detected for selective organelles and compartments should be mainly derived from the well-controlled selective proteolysis processes, instead of the lysis solvent and less-selective lysosome-involved proteolyses. We plan to explore the use of hydrophobic solvents for extension of the method to study peptides containing hydrophobic sequences, e.g., membrane domain peptides and signal peptides.

One might predict that the yeast cytoplasm would be the most active location for proteolytic breakdown of proteins for cells that experienced a nutrient-rich, rapid growth environment at the time of harvest, as proteasomes are mainly distributed in the yeast cytoplasm. We did observe the proteasomal trypsin-like and chymotrypsin-like activities for the breakdown of yeast cytoplasmic proteins, but found less evidences for the caspase-like activity. From the terminal amino acids of the peptides detected, we observed the steric preference for proteolytic degradation of yeast cytoplasmic proteins. This steric preference could be related to a strict requirement for protein proteolysis to occur in proteasomes, but is less necessary for protein breakdown by lysosomal proteinases (see Supplementary Notes). Further investigation (e.g., using an inhibitor to control specific degradation pathways) is needed to better understand the mechanism of protein proteasomal degradation. Our data also indicates that the cytoplasmic proteasomes could also be responsible for the degradation of the ATPase V₁ domain proteins after their disassembly and most of the multiple organelle-co-localized proteins observed in this study (e.g., 83 of the 87 multiple organelle-co-localized proteins assigned are associated as being localized to the cytoplasm).

Whether the yeast nucleus is proteolysis-active or not has not been clearly elucidated yet, and few experimental data have reported the proteolysis activities happening in the yeast nucleus. Proteasomes (>100 Å in size) cannot be directly imported into the yeast nucleus through the smaller nucleus pores (<90 Å for free diffusion) in the envelope, but the proteasome subunit complexes have been reported to be imported into yeast nucleus as precursor complexes.³⁰ Existence of proteasome precursor complexes in the yeast nucleus, however, cannot necessarily be concluded since after importation, these complexes would have to be assembled to form the matured, proteolysis-active proteasomes. Our results provide evidence to the contrary, at least for the growth condition we explored. Further studies involving the isolation of the nucleus could help to determine if there is indeed proteolytic activity occurring in the nucleus of yeast.

Ubiquitylation is a well-established pathway for protein proteasomal degradation, and almost all ribosomal and stress-response proteins identified in this study by the detection of the intermediate peptides were ubiquitylation proteins (http://ubiprot.org.ru/). However, an overwhelming majority of cytoplasmic regulatory and enzyme proteins that produced the degradation intermediate peptides identified are not associated as being ubiquitylation proteins (see Table 1). The documented ubiquitylation protein list may be incomplete, but this is unlikely the reason for our lack in comprehensive observation of these proteins as the possibility for the measurements of protein ubiquitylation should be the same for ribosomal & stress-response proteins and regulatory & enzyme proteins with consideration that these different proteins from different functional groups have a similar range of lifetime span and expression levels. From our results of the proteome-wide measurements of intracellular peptides, we speculate that some non-ubiquitylation-proteasome pathways⁴⁸^,⁴⁹ or even undefined proteolytic processes that need further study were also possibly functioning in the yeast cytoplasm which was responsible for the observation of degraded regulatory and enzyme proteins.

Mass spectrometry-based methods have been widely used for various proteomics applications. Information of protein post-translational degradation, however, has been generally understudied or totally ignored in most analyses attempting to elucidate protein expressions and functions in the cell biological processes involved. Since degradation events are ever present in cells and are as biologically important as protein creation, more emphasis needs to take place in the field of proteomics on the understanding of the simultaneous protein breakdown occurring. Developments in liquid phase separations, mass spectrometers, and informatics now allows for reliable assessment of proteome-wide protein degradation and can provide proteomics with additional information of protein degradation/turnover for more precise elucidation of protein expressions and cellular function. Finally, in combination with the control of some cellular process (e.g., with use of some inhibitors) the specific pathways of protein degradation and the related diseases⁵⁰^,⁵¹ could be more directly, in a high-throughput fashion, studied with the method described here for better understanding of what is taking place within the cell than with the traditional approaches currently used in proteomics and protein degradation studies.

METHODS

Preparation of soluble yeast lysate

Saccharomycess cerevisiae (ATCC 90845) was grown in a batch shaker flask at 30°C on YPD broth. Cells were harvested by centrifugation at 4,000 rpm for 10 min at mid-logarithmic phase. Cells were washed twice with 4°C phosphate buffered saline, pH 7.4 containing a protease inhibitor cocktail (complete, mini tablet) at the concentration recommended by the manufacturer (Roche Applied Science, Mannheim Germany). The cells were washed by adding 2×the volume of buffer described above to the cell pellet followed with a gentle vortex and a repeat of the centrifugation followed by a decantation of the supernatant. After the second the supernatant was decanted from the cell pellet and immediately the yeast cells were flash frozen with liquid N₂ and stored at −80°C until ready for further processing. To a 200 µL volume sized cell pellet of yeast Saccharomyces cerevisiae, 1.3 mL of 18.3 MΩ water containing a protease inhibitor cocktail (complete, mini tablet) at the concentration recommended by the manufacturer (Roche Applied Science) was added and the suspension was vortexed. Lysis was achieved using pressure cycling technology capable with the Barocycler™ (Pressure BioSciences, West Bridgewater, MA) for 10 cycles going between ambient pressure for 20 sec and 35000 psi for 20 sec at 4°C. The lysate was ultracentrifuged at 100,000 rpm (355,040 rcf) with a Beckman (Fullerton, CA) Optima TL ultracentrifuge at 4°C for 20 min. The supernatant was collected and placed immediately on ice. A BCA protein assay (Pierce, Rockford IL) was performed to determine the protein concentration using a bovine serum albumin standard. The sample was flash frozen with liquid N₂.

High-resolution LC-high-precision MS/MS experiments

The prepared lysate was analyzed using high-resolution LC-high-precision MS/MS as follows. The high-resolution LC was performed on an LC instrument as previously reported.⁵² A 120 cm × 100 µm i.d. fused silica capillary column containing 3-µm (120 Å size) C4-silica particles (Phenomenex, Terrence, CA) was used as the LC column. The sample (1 µg/µL protein content) was loaded into a 10 µL loop, switched onto the LC column, and separated with a gradient from mobile phase A acetonitrile/H₂O/acetic acid (25:75:0.2, v/v/v) to mobile phase B acetonitrile/H₂O/acetic acid/trifluroacetic acid (90:10:0.2:0.1, v/v/v/v) at room temperature. (The use of 25% ACN in mobile phase A was to prevent precipitation of proteins existing in the lysate and the use of TFA in the mobile phase B was to improve elution of various sizes and properties of peptides/proteins.) The separation was completed in 600 min at an operation pressure of 15K psi; the reproducibility of such a long high-resolution LC coupled with the mass spectrometers has been systematically evaluated⁵² and no repeated runs were made herein. The high-precision MS/MS was performed on an LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific., San Jose, CA). FT-MS and FT-MS/MS data were collected with AGC targets of 1 × 10⁶ and 2 × 10⁵, respectively. Spectra were acquired at a 30K resolution, using a survey scan with 400≤m/z≤2000 followed by FT-MS/MS of the 5 most intense ions from the survey scan (monoisotopic precursor selection was not enabled). F T-MS/MS employed an isolation window of 3 m/z units and 35% normalized collision energy. Dynamic exclusion was enabled with no repeat counts using a mass window of ±1.5 m/z units and a duration cycle of 25 sec. Mass calibration was performed according to the method provided by the instrument manufacturer.

Analysis of high-precision MS/MS data

The previously described method⁵³ was used to process the high-precision MS/MS data. Briefly, the FT MS/MS data were initially processed using SEQUEST (Sequest 27_master, rev 12, Thermo Fisher Scientific) with molecular mass tolerances of ±5 u and ±210 u in two database searches, respectively. The amino acid sequences available from the high-precision MS/MS spectra were recalculated for the SEQUEST-recommended top 10 peptide candidates using ICR2LS software developed in-house for analysis of mass spectrometry data (http://ncrr.pnl.gov/software/ICR2LS.stm; the ICR2LS parameter settings used in this study are detailed in Supplementary Notes). The theoretical isotopic envelopes for the fragments of each peptide candidate and its precursor were generated by use of ICR2LS. For the peptide candidate fragments, we matched the b and y fragments predicted from the candidates against the de-isotoped monoisotopic masses (i.e., deconvolved isotopic envelopes) of the spectrum using ICR2LS. If the match was achieved with a mass error tolerance of 10 ppm, the b and y fragments were accepted as assigned. The consecutive b or y fragments were used to construct the amino acid sequence(s). During construction of amino acid sequence(s), we required that each sequence must contain at least 3 fragments that had the isotopic-envelopes (≥2 isotopic peaks) matched with theoretical ones within the 10-ppm mass tolerance. The constructed sequences from all MS/MS spectra were then searched against the yeast protein database to examine the sequence uniqueness with consideration of influence from various possible modifications,⁵³ and only the unambiguous unique sequences (referred as unique sequence tags, UStags) were used for identification of the peptides. For each UStag-identified peptide, we matched its theoretical isotopic envelope to that of the corresponding precursor that we required to have at least 3 most abundant isotopic peaks for matching. If the match was within a mass error tolerance of 10 ppm, the peptide was considered to be identified without modification. If the mass error was larger than 10 ppm, we assumed existence of some modifications. According to the mass difference between the peptide identified and its precursor, we examined the spectra (including with manual inspection by use of XCalibur 2.0.5 and ICR2LS) to determinate the modifications. At this step, we simultaneously corrected the charge states wrongly assigned by SEQUEST (Supplementary Table 3). The mass errors for various types of matching, including molecular mass and averaged values for fragmental mass and amino acid residues, are given in the identification list (Supplementary Table 1). The dataset search results for the identified peptides are given in Supplementary Table 3 for additional information, and 20 spectra randomly selected for various charge states were given in Supplementary Table 4.

In this study, the hydrophobity GRAVY values were calculated according to the method reported in ref. ⁵⁴.

Supplementary Material

Notes1

Supplementary notes, figures, and tables were attached with this manuscript.

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Table1

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Table3

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Table4

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ACKNOWLEDGMENTS

We thank Dr. Robert A. Maxwell for his informational discussion. This research was partially supported by the U.S. Department of Energy (DOE) Office of Biological and Environmental Research, the NIH National Center for Research Resources (RR18522), and the Environmental Molecular Science Laboratory, a DOE national scientific user facility located on the campus of Pacific Northwest National Laboratory (PNNL) in Richland, Washington. PNNL is a multi-program national laboratory operated by Battelle Memorial Institute for the DOE under contract DE-AC05-76RLO-1830.

References

1. Goldberg AL. Protein degradation and protection against misfolded or damaged peoteins. Nature. 2003;426:895–899. [PubMed]

2. Demartino GN, Gillette TG. Proteasomes: Machines for all reasons. Cell. 2007;129:659–662. [PubMed]

3. Lee DH, Sherman MY, Goldberg AL. Involvement of the molecular chaperone Ydj1 in the ubiquitin-dependent degradation of short lived and abnormal proteins in Saccharomyces cerevisiae. Mol. Cell. Biol. 1996;16:4773–4781. [PMC free article] [PubMed]

4. Goldberg AL, Rock KL. Proteolysis, proteasomes and antigen presentation. Nature. 1992;357:375–379. [PubMed]

5. Baumeister W, Walz J, Zühl F, Seemüller E. The proteasome: paradigram of a self-compartmentalizing protease. Cell. 1998;92:367–380. [PubMed]

6. Wenzel T, Eckerskorn C, Lottspeich F, Baumeister W. Existence of a molecular ruler in proteasomes suggested by analysis of dergreadation products. FEBS Lett. 1994;349:205–209. [PubMed]

7. Kisselev AF, Akopian TN, Woo KM, Goldberg AL. The size of peptides generated from protein by mammalian 26 and 20S proteasomes. J. Biol. Chem. 1999;274:3363–3371. [PubMed]

8. Hershko A, Ciechanover A. The ubiquitin system. Annu. Rev. Biochem. 1998;67:425–479. [PubMed]

9. Weissman AM. Themes and variations on uniquitylation. Nat. Rev. Mol. Cell Biol. 2001;2:169–178. [PubMed]

10. Welchman RL, Cordon C, Mayer RJ. Ubiquitin and ubiquitin-like proteins as multifunctional signals. Nat. Rev. Mol. Cell Biol. 2005;6:599–609. [PubMed]

11. Scaric T, Graef CI, Goldberg AL. Pathway for degradation of peptides generated by proteasomes. J. Biol. Chem. 2004;279:46723–46732. [PubMed]

12. Rubinsztein DC. The roles of intracellular protein-degradation pathways in neurodegradation. Nature. 2006;443:780–786. [PubMed]

13. Klionsky DJ, Emr SD. Autophagy as a regulated pathway of cellular degradation. Science. 2000;290:1717–1721. [PMC free article] [PubMed]

14. Yoshimori T. Autophagy: paying Charon’s toll. Cell. 2007;128:833–836. [PubMed]

15. Knop M, Schiffer HH, Pupp S, Wolf DH. Vacuolar/lysosomal proteolysis: proteases, substrates, mechanisms. Curr. Opin. Cell. Biol. 1993;5:990–996. [PubMed]

16. Jones EW. Vacuolar proteases and proteolytic artifacts in Saccharomyces cerevisiae. Methods Enzymol. 2002;351:127–150. [PubMed]

17. Rendueles PS, Wolf DH. Proteinase function in yeast: biochemical and genetic approaches to a central mechanism of post-translational control in the eukaryote cell. FEMS Microbiol. Rev. 1988;4:17–45. [PubMed]

18. Falk K, Rötzschke O, Rammensee HG. Cellular peptide composition governed by major histocompatibility complex class I molecules. Nature. 1990;348:248–251. [PubMed]

19. Reits E, et al. Peptide diffusion, protection, and degradation in nuclear and cytoplasmic compartments before antigen presentation by MHC class I. Immunity. 2003;18:97–108. [PubMed]

20. Huh W-K, et al. Global analysis of protein localization in budding yeast. Nature. 2003;425:686–691. [PubMed]

21. Ghaemmaghami S, et al. Global analysis of protein expression in yeast. Nature. 2003;425:737–741. [PubMed]

22. Belle A, Tanay A, Bitincka L, Shamir R, O’Shea EK. Quantification of protein half-lives in the budding yeast. Proc. Natl. Acad. Sci. USA. 2006;103:13004–13009. [PubMed]

23. Bendtsen JD, Nielsen H, von Heijne G, Brunak S. Improved prediction of signal peptides: SignalP 3.0. J. Mol. Biol. 2004;340:783–795. [PubMed]

24. Rockwell NC, Krysan DJ, Komiyama T, Fuller RS. Precursor processing by Kex2/furin proteases. Chem. Rev. 2002;102:4525–4548. [PubMed]

25. Nussbaum AK, et al. Cleavage motifs of the yeast 20S proteasome β subunits deduced from digests of enolase 1. Proc. Natl. Acad. Sci. USA. 1998;95:12504–12509. [PubMed]

26. Li X, Chang YH. Amino-terminal protein processing in Saccharomyces cerevisiae is an essential function that requires two distinct methionine aminopeptidases. Proc. Natl. Acad. Sci. USA. 1995;92:12357–12361. [PubMed]

27. Piwko W, Jentsch S. Proteasome-mediated proteins processing by bidirectional degradation initiated from an internal site. Nat. Struc.t Mo.l Biol. 2006;13:691–697. [PubMed]

28. Löwe L, et al. Crystal structure of the 20S proteasome from the archaeon T. acidophilum at 3.4Å resolution. Science. 1995;268:533–539. [PubMed]

29. Niedermann G, et al. The proteolytic fragments generated by vertebrate proteasomes: structural relationships to major histocompatibility complex class I binding peptides. Proc. Natl. Acad. Sci. USA. 1996;93:8572–8577. [PubMed]

30. Lehmann A, Janek K, Braun B, Kloetzel P-K, Enenkel C. 20 S proteasomes are imported as precursor complexes into the nucleus of yeast. J. Mol. Biol. 2002;317:401–413. [PubMed]

31. Fahrenkrog B, Aris JP, Hurt EC, Panté N, Aebi U. Comparative spatial localization of protein-A-tagged and authentic yeast nuclear pore complex proteins by immunogold electron microscopy. J. Struct. Biol. 2000;129:295–305. [PubMed]

32. Matsuura Y, Lange A, Harreman MT, Corbett AH, Stewart M. Structural basis for Nup2P function in cargo release and karyopherin recycling in nuclear import. EMBO J. 2003;22:5358–5369. [PubMed]

33. Finbow ME, Harrison MA. The vacuolar H⁺-ATPase: a universal proteom pump of eukaryotes. Biochem. J. 1997;324:697–712. [PubMed]

34. Holliday LS, et al. The amino-terminal domain of the B subunit of vacuolar H+-ATPase contains a filamentous actin binding site. J. Biol. Chem. 2000;275:32331–32337. [PubMed]

35. Bota DA, Davies KJA. Protein degradation in mitochondria: implications for oxidative stress, aging and disease: a novel etiological classification of mitochondrial proteolytic disorders. Mitochondrion. 2001;1:33–49. [PubMed]

36. Ohba M, Schatz G. Protein import into yeast mitochondria is inhibited by antibodies raised against 45-kd proteins of the out membrane. EMBO J. 1987;6:2109–2115. [PubMed]

37. Van der Laan M, Rissler M, Rehling P. Mitochondrial preprotein translocases as dynamic molecular machines. FEMS Yeast Res. 2006;6:849–861. [PubMed]

38. Theriot JA. Accelerating on a treadmill: ADF/cofilin promotes rapid actin filament turnover in the dynamic cytoskeleton. J. Cell Biol. 1997;136:1165–1168. [PMC free article] [PubMed]

39. Hubberstey AV, Mottillo EP. Cyclase-associated proteins: Capacity for linking signal transduction and actin polymerization. FASEB J. 2002;16:487–499. [PubMed]

40. Kassenbrock CK, Garcia PD, Walter P, Kelly RB. Heavy-chain binding protein recognizes aberrant polypeptides translocated in vitro. Nature. 1988;333:90–93. [PubMed]

41. Jackson CJ, Képès F. BFR1, a multicopy suppressor of bredeldin A-induced lethality, is implicated in secretion and nuclear segregation in Saccharomyces cerevisiae. Genetics. 1994;137:423–437. [PubMed]

42. Washburn MP, Wolters D, Yates JR. Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 2001;19:242–247. [PubMed]

43. Svensson M, et al. Neuropeptidomics: expanding proteomics downwards. Biochem Soc Trans. 2007;35:588–593. [PubMed]

44. López-Otín C, Overall CM. Protease degradomics: a new challenge for proteomics. Nat. Rev. Mol. Cell Biol. 2002;3:509–519. [PubMed]

45. Smejkal GB, et al. Sample preparation for two-dimensional gel electrophoresis using pressure cycling technology. Anal. Biochem. 2007;363:309–311. [PubMed]

46. Katrusiak A, McMillan P, editors. High-pressure crystallography; Proceedings of the NATO advanced research workshop on high-pressure crystallography; Italy: Erice; 2003. pp. 543–556.

47. Shen Y, et al. High-efficiency on-line solid-phase extraction coupling to 15–150-µm-i. d. column liquid chromatography for proteomic analysis. Anal. Chem. 2003;75:3596–3605. [PubMed]

48. Murakami Y, et al. Ornithine decarboxylase is degraded by the 26S proteasome without ubiquitination. Nature. 1992;360:597–599. [PubMed]

49. Jariel-Encontre I, et al. Ubiquitylation is not an absolute requirement for degradation of c-Jun protein by the 26S proteasome. J. Biol. Chem. 1996;270:11623–11627. [PubMed]

50. Reinstein E, Ciechanover A. Narrative review: protein degradation and human diseases: the ubiquitin concerns. Ann. Intern. Med. 2006;145:676–684. [PubMed]

51. Meijer AJ, Codogno P. Signalling and autophagy regulation in health, aging and diseases. Mol. Aspects Med. 2006;27:411–425. [PubMed]

52. Shen Y, et al. Automated 20 kpsi RPLC-MS and MS/MS with chromatographic peak capacities of 1000–1500 and capabilities in proteomics and metabolomics. Anal. Chem. 2005;77:3090–3100. [PubMed]

53. Shen Y, et al. Proteome-wide identification of proteins and their modifications with decreased ambiguities and improved false discovery rates using unique sequence tags. Anal. Chem. 2008;80:1871–1882. [PMC free article] [PubMed]

54. Kyte J, Doolittle RF. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 1982;157:105–132. [PubMed]