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 ). 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 ).
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 (). (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 , , and ). 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 ). 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.