Enriching Cysteine-Containing Peptides Using a Sulfhydryl-Reactive Alkylating Reagent with a Phosphonic Acid Group and Immobilized Metal Affinity Chromatography.

The reduction of disulfide bonds and their subsequent alkylation are commonplace in typical proteomics workflows. Here, we highlight a sulfhydryl-reactive alkylating reagent with a phosphonic acid group (iodoacetamido-LC-phosphonic acid, 6C-CysPAT) that facilitates the enrichment of cysteine-containing peptides for isobaric tag-based proteome abundance profiling. Specifically, we profile the proteome of the SH-SY5Y human cell line following 24 h treatments with two proteasome inhibitors (bortezomib and MG-132) in a tandem mass tag (TMT)pro9-plex experiment. We acquire three datasets─(1) Cys-peptide enriched, (2) the unbound complement, and (3) the non-depleted control─and compare the peptides and proteins quantified in each dataset, with emphasis on Cys-containing peptides. The data show that enrichment using 6C-Cys phosphonate adaptable tag (6C-CysPAT) can quantify over 38,000 Cys-containing peptides in 5 h with >90% specificity. In addition, our combined dataset provides the research community with a resource of over 9900 protein abundance profiles exhibiting the effects of two different proteasome inhibitors. Overall, the seamless incorporation of alkylation by 6C-CysPAT into a current TMT-based workflow permits the enrichment of a Cys-containing peptide subproteome. The acquisition of this "mini-Cys" dataset can be used to preview and assess the quality of a deep, fractionated dataset.

Enhancing Proteome Coverage by Using Strong Anion-Exchange in Tandem with Basic-pH Reversed-Phase Chromatography for Sample Multiplexing-Based Proteomics.

Sample multiplexing-based proteomic strategies rely on fractionation to improve proteome coverage. Tandem mass tag (TMT) experiments, for example, can currently accommodate up to 18 samples with proteins spanning several orders of magnitude, thus necessitating fractionation to achieve reasonable proteome coverage. Here, we present a simple yet effective peptide fractionation strategy that partitions a pooled TMT sample with a two-step elution using a strong anion-exchange (SAX) spin column prior to gradient-based basic pH reversed-phase (BPRP) fractionation. We highlight our strategy with a TMTpro18-plex experiment using nine diverse human cell lines in biological duplicate. We collected three data sets, one using only BPRP fractionation and two others of each SAX-partition followed by BPRP. The three data sets quantified a similar number of proteins and peptides, and the data highlight noticeable differences in the distribution of peptide charge and isoelectric point between the SAX partitions. The combined SAX partition data set contributed 10% more proteins and 20% more unique peptides that were not quantified by BPRP fractionation alone. In addition to this improved fractionation strategy, we provide an online resource of relative abundance profiles for over 11,000 proteins across the nine human cell lines, as well as two additional experiments using ovarian and pancreatic cancer cell lines.

Nuclear PP2A-Cdc55 prevents APC-Cdc20 activation during the spindle assembly checkpoint.

Cdc55, a regulatory B-subunit of protein phosphatase 2A (PP2A) complex, is essential for the spindle assembly checkpoint (SAC) in budding yeast, but the regulation and molecular targets of PP2A-Cdc55 have not been clearly defined or are controversial. Here, we show that an important target of Cdc55 in the SAC is the anaphase-promoting complex (APC) coupled with Cdc20 and that APC-Cdc20 is kept inactive by dephosphorylation by nuclear PP2A-Cdc55 when spindle is damaged. By isolating a new class of Cdc55 mutants specifically defective in the SAC and by artificially manipulating nucleocytoplasmic distribution of Cdc55, we further show that nuclear Cdc55 is essential for the SAC. Because the Cdc55-binding proteins Zds1 and Zds2 inhibit both nuclear accumulation of Cdc55 and SAC activity, we propose that spatial control of PP2A by Zds1 family proteins is important for tight control of SAC and mitotic progression.

A yeast GSK-3 kinase Mck1 promotes Cdc6 degradation to inhibit DNA re-replication.

Cdc6p is an essential component of the pre-replicative complex (pre-RC), which binds to DNA replication origins to promote initiation of DNA replication. Only once per cell cycle does DNA replication take place. After initiation, the pre-RC components are disassembled in order to prevent re-replication. It has been shown that the N-terminal region of Cdc6p is targeted for degradation after phosphorylation by Cyclin Dependent Kinase (CDK). Here we show that Mck1p, a yeast homologue of GSK-3 kinase, is also required for Cdc6 degradation through a distinct mechanism. Cdc6 is an unstable protein and is accumulated in the nucleus only during G1 and early S-phase in wild-type cells. In mck1 deletion cells, CDC6p is stabilized and accumulates in the nucleus even in late S phase and mitosis. Overexpression of Mck1p induces rapid Cdc6p degradation in a manner dependent on Threonine-368, a GSK-3 phosphorylation consensus site, and SCF(CDC4). We show evidence that Mck1p-dependent degradation of Cdc6 is required for prevention of DNA re-replication. Loss of Mck1 activity results in synthetic lethality with other pre-RC mutants previously implicated in re-replication control, and these double mutant strains over-replicate DNA within a single cell cycle. These results suggest that a GSK3 family protein plays an unexpected role in preventing DNA over-replication through Cdc6 degradation in Saccharomyces cerevisiae. We propose that both CDK and Mck1 kinases are required for Cdc6 degradation to ensure a tight control of DNA replication.

Specificity profiling of deubiquitylases against endogenously generated ubiquitin-protein conjugates.

Deubiquitylating enzymes (DUBs) remove ubiquitin from proteins thereby regulating their stability or activity. Our understanding of DUB-substrate specificity is limited because DUBs are typically not compared to each other against many physiological substrates. By broadly inhibiting DUBs in Xenopus egg extract, we generated hundreds of ubiquitylated proteins and compared the ability of 30 DUBs to deubiquitylate them using quantitative proteomics. We identified five high-impact DUBs (USP7, USP9X, USP36, USP15, and USP24) that each reduced ubiquitylation of over 10% of the isolated proteins. Candidate substrates of high-impact DUBs showed substantial overlap and were enriched for disordered regions, suggesting this feature may promote substrate recognition. Other DUBs showed lower impact and non-overlapping specificity, targeting distinct non-disordered proteins including complexes such as the ribosome or the proteasome. Altogether our study identifies candidate DUB substrates and defines patterns of functional redundancy and specificity, revealing substrate characteristics that may influence DUB-substrate recognition.

Comparison of the Proteomes and Phosphoproteomes of S. cerevisiae Cells Harvested with Different Strategies.

The budding yeast Saccharomyces cerevisiae is a powerful model system that is widely used to investigate many cellular processes. The harvesting of yeast cells is the first step in almost every experimental procedure. Here, yeast cells are isolated from their growth medium, collected, and used for successive experiments or analysis. The two most common methods to harvest S. cerevisiae are centrifugation and filtration. Understanding if and how centrifugation and filtration affect yeast physiology is essential with respect to downstream data interpretation. Here, we profile and compare the proteomes and the phosphoproteomes, using isobaric label-based quantitative mass spectrometry, of three common methods used to harvest S. cerevisiae cells: low-speed centrifugation, high-speed centrifugation, and filtration. Our data suggest that, while the proteome was stable across the tested conditions, hundreds of phosphorylation events were different between centrifugation and filtration. Our analysis shows that, under our experimental conditions, filtration may cause both cell wall and osmotic stress at higher levels compared to centrifugation, implying harvesting-method-specific stresses. Thus, considering that the basal activation levels of specific stresses may differ under certain harvesting conditions is an important, but often overlooked, aspect of experimental design.

Spin column-based peptide fractionation alternatives for streamlined tandem mass tag (SL-TMT) sample processing.

Fractionation is essential to achieving deep proteome coverage for sample multiplexing experiments where currently up to 18 samples can be analyzed concurrently. However, peptide fractionation (i.e., upstream of LC-MS/MS analysis) with a liquid chromatography system constrains sample processing as only a single sample can be fractionated at once. Here, we highlight the use of spin column-based methods which permit multiple multiplexed samples to be fractionated simultaneously. These methods require only a centrifuge and eliminate the need for a dedicated liquid chromatography system. We investigate peptide fractionation with strong anion exchange (SAX) and high-pH reversed phase (HPRP) spin columns, as well as a combination of both. In two separate experiments, we acquired deep proteome coverage (>8000 quantified proteins), while starting with <25 µg of protein per channel. Our datasets showcase the proteome alterations in two human cell lines resulting from treatment with inhibitors acting on the ubiquitin-proteasome system. We recommend this spin column-based peptide fractionation strategy for high-throughput screening applications or whenever a liquid chromatograph is not readily available. SIGNIFICANCE: Fractionation is a means to achieve deep proteome coverage for global proteomics analysis. Typical liquid chromatography systems may be a prohibitive expense for many laboratories. Here, we investigate prefractionation with strong anion exchange (SAX) and high-pH reversed phase (HPRP) spin columns, as well as a combination of both, as peptide fractionation methods. These spin columns have advantages over liquid chromatography systems, which include relative affordability, higher throughput capability, no carry over, and fewer potential instrument-related malfunctions. In two separate experiments, we acquired deep proteome coverage (>8000 quantified proteins), thereby showing the utility of each or a combination of both spin columns for global proteome analysis.

Identification of Deubiquitinase Substrates in Xenopus Egg Extract.

Deubiquitinases (DUBs) antagonize protein ubiquitination by removing ubiquitin from substrates. Identifying the physiological substrates of each DUB is critical for understanding DUB function and the principles that govern the specificity of this class of enzymes. Since multiple DUBs can act on the same substrate, it can be challenging to identify substrates using inactivating a single enzyme. Here, we outline a method that enables the identification of proteins whose stability depends on DUB activity and an approach to profile DUB specificity in Xenopus egg extract. By coupling broad DUB inhibition with quantitative proteomics, we circumvent DUB redundancy to identify DUB substrates. By adding back recombinant DUBs individually to the extract, we pinpoint DUBs sufficient to counteract proteasomal degradation of these newly identified substrates. We apply this method to Xenopus egg extract but suggest that it can also be adapted to other cell lysates.

Comparative Proteomic Analysis of Two Commonly Used Laboratory Yeast Strains: W303 and BY4742.

The yeast Saccharomyces cerevisiae is a powerful model system that is often used to expand our understanding of cellular processes and biological functions. Although many genetically well-characterized laboratory strains of S. cerevisiae are available, they may have different genetic backgrounds which can confound data interpretation. Here, we report a comparative whole-proteome analysis of two common laboratory yeast background strains, W303 and BY4742, in both exponential and stationary growth phases using isobaric-tag-based mass spectrometry to highlight differences in proteome complexity. We quantified over 4400 proteins, hundreds of which showed differences in abundance between strains and/or growth phases. Moreover, we used proteome-wide protein abundance to profile the mating type of the strains used in the experiment, the auxotrophic markers, and associated metabolic pathways, as well as to investigate differences in particular classes of proteins, such as the pleiotropic drug resistance (PDR) proteins. This study is a valuable resource that offers insight into mechanistic differences between two common yeast background strains and can be used as a guide to select a background that is best suited for addressing a particular biological question.

Substrate identification and specificity profiling of deubiquitylases against endogenously-generated ubiquitin-protein conjugates.

Deubiquitylating enzymes (DUBs) remove ubiquitin from proteins thereby regulating their stability or activity. Our understanding of DUB-substrate specificity is limited because DUBs are typically not compared to each other against many physiological substrates. By broadly inhibiting DUBs in Xenopus egg extract, we generated hundreds of ubiquitylated proteins and compared the ability of 30 DUBs to deubiquitylate them using quantitative proteomics. We identified five high impact DUBs (USP7, USP9X, USP36, USP15 and USP24) that each reduced ubiquitylation of over ten percent of the isolated proteins. Candidate substrates of high impact DUBs showed substantial overlap and were enriched for disordered regions, suggesting this feature may promote substrate recognition. Other DUBs showed lower impact and non-overlapping specificity, targeting distinct non-disordered proteins including complexes such as the ribosome or the proteasome. Altogether our study identifies candidate DUB substrates and defines patterns of functional redundancy and specificity, revealing substrate characteristics that may influence DUB-substrate recognition.

Proteome-Wide Profiling Using Sample Multiplexing of a Human Cell Line Treated with Cannabidiol (CBD) and Tetrahydrocannabinol (THC).

Cannabis has been used historically for both medicinal and recreational purposes, with the most notable cannabinoids being cannabidiol (CBD) and tetrahydrocannabinol (THC). Although their therapeutic effects have been well studied and their recreational use is highly debated, the underlying mechanisms of their biological effects remain poorly defined. In this study, we use isobaric tag-based sample multiplexed proteome profiling to investigate protein abundance differences in the human neuroblastoma SH-SY5Y cell line treated with CBD and THC. We identified significantly regulated proteins by each treatment and performed a pathway classification and associated protein-protein interaction analysis. Our findings suggest that these treatments may lead to mitochondrial dysfunction and induce endoplasmic reticulum stress. These data can potentially be interrogated further to investigate the potential role of CBD and THC in various biological and disease contexts, providing a foundation for future studies.

The budding yeast PP2ACdc55 protein phosphatase prevents the onset of anaphase in response to morphogenetic defects.

Faithful chromosome transmission requires establishment of sister chromatid cohesion during S phase, followed by its removal at anaphase onset. Sister chromatids are tethered together by cohesin, which is displaced from chromosomes through cleavage of its Mcd1 subunit by the separase protease. Separase is in turn inhibited, up to this moment, by securin. Budding yeast cells respond to morphogenetic defects by a transient arrest in G2 with high securin levels and unseparated chromatids. We show that neither securin elimination nor forced cohesin cleavage is sufficient for anaphase in these conditions, suggesting that other factors contribute to cohesion maintainance in G2. We find that the protein phosphatase PP2A bound to its regulatory subunit Cdc55 plays a key role in this process, uncovering a new function for PP2A(Cdc55) in controlling a noncanonical pathway of chromatid cohesion removal.

Quantitative proteome and phosphoproteome datasets of DNA replication and mitosis in Saccharomyces cerevisiae.

Cell division is a highly regulated process that secures the generation of healthy progeny in all organisms, from yeast to human. Dysregulation of this process can lead to uncontrolled cell proliferation and genomic instability, both which are hallmarks of cancer. Cell cycle progression is dictated by a complex network of kinases and phosphatases. These enzymes act on their substrates in a highly specific temporal manner ensuring that the process of cell division is unidirectional and irreversible. Key events of the cell cycle, such as duplication of genetic material and its redistribution to daughter cells, occur in S-phase and mitosis, respectively. Deciphering the dynamics of phosphorylation/dephosphorylation events during these cell cycle phases is important. Here, we showcase a quantitative proteomic and phosphoproteomic mass spectrometry dataset that profiles both early and late phosphorylation events and associated proteome alterations that occur during S-phase and mitotic arrest in the model organism S. cerevisiae. This dataset is of broad interest as the molecular mechanisms governing cell cycle progression are conserved throughout evolution. The data has been deposited in ProteomeXchange with the dataset identifier PXD037291.

Quantitative proteome dataset profiling of UBC4 and UBC5 deletion strains in Saccharomyces cerevisiae.

The Ubiquitin-Proteasome System (UPS) regulates many cellular processes in eukaryotic cells. Ubiquitylation by the UPS mainly directs proteins to proteasomal degradation, but it can also have non-degradative functions, such as regulating protein activity or localization. The small protein ubiquitin is conjugated to its substrates via a cascade of E1-E2-E3 enzymes. Dysregulation of the UPS has been implicated in the genesis and progression of many diseases, such as neurodegenerative diseases and cancer; thus, the UPS components are attractive targets for developing pharmaceutical drugs. E2s, or ubiquitin conjugating enzymes, are central players of the UPS. E2s function in tandem with specific ubiquitin ligases (E3s) to transfer ubiquitin to substrates. Here, we present the first proteome stability analysis of two closely related ubiquitin conjugating enzymes, Ubc4 and Ubc5, in S. cerevisiae. These two E2s are nearly identical, having 92% sequence identity and differing by only 11 amino acid residues. This dataset is of broad interest because higher eukaryotes express ubiquitin conjugating enzymes that are analogous to the yeast Ubc4/5. The data have been deposited in ProteomeXchange with the dataset identifier PXD037315.

Fe3+-NTA magnetic beads as an alternative to spin column-based phosphopeptide enrichment.

Protein phosphorylation is a central mechanism of cellular signal transduction in living organisms. Phosphoproteomic studies systematically catalogue and characterize alterations in phosphorylation states across multiple cellular conditions and are often incorporated into global proteomics experiments. Previously, we found that spin column-based Fe3+-NTA enrichment integrated well with our workflow but remained a bottleneck for methods that require higher throughput or a scale that is beyond the capacity of these columns. Here, we compare our well-established spin column-based enrichment strategy with one encompassing magnetic beads. Our data show little difference when using either method in terms of the number of identified phosphopeptides as well as their physicochemical properties. In all, we illustrate how the potentially scalable and automation-friendly magnetic Fe3+-NTA beads can seamlessly substitute spin column-based Fe3+-NTA agarose beads for global phosphoproteome profiling. SIGNIFICANCE: Protein phosphorylation plays a key role in regulating a multitude of biological processes and can lead to insights into disease pathogenesis. Methodologies which can efficiently enrich phosphopeptides in a scalable and high-throughput manner are essential for profiling dynamic phosphoproteomes. Here we compare two phosphopeptide enrichment workflows, a well-established spin column-based strategy with agarose Fe3+-NTA beads and a strategy using magnetic Fe3+-NTA beads. Our data suggest that the scalable and automation-friendly magnetic bead-based workflow is an equivalent, but more flexible, enrichment strategy for phosphoproteome profiling experiments.

Proteomics of broad deubiquitylase inhibition unmasks redundant enzyme function to reveal substrates and assess enzyme specificity.

Deubiquitylating enzymes (DUBs) counteract ubiquitylation to control stability or activity of substrates. Identification of DUB substrates is challenging because multiple DUBs can act on the same substrate, thwarting genetic approaches. Here, we circumvent redundancy by chemically inhibiting multiple DUBs simultaneously in Xenopus egg extract. We used quantitative mass spectrometry to identify proteins whose ubiquitylation or stability is altered by broad DUB inhibition, and confirmed their DUB-dependent regulation with human orthologs, demonstrating evolutionary conservation. We next extended this method to profile DUB specificity. By adding recombinant DUBs to extract where DUB activity was broadly inhibited, but ubiquitylation and degradation were active at physiological rates, we profiled the ability of DUBs to rescue degradation of these substrates. We found that USP7 has a unique ability to broadly antagonize degradation. Together, we present an approach to identify DUB substrates and characterize DUB specificity that overcomes challenges posed by DUB redundancy.

Adapt or die: how eukaryotic cells respond to prolonged activation of the spindle assembly checkpoint.

Many cancer-treating compounds used in chemotherapies, the so-called antimitotics, target the mitotic spindle. Spindle defects in turn trigger activation of the SAC (spindle assembly checkpoint), a surveillance mechanism that transiently arrests cells in mitosis to provide the time for error correction. When the SAC is satisfied, it is silenced. However, after a variable amount of time, cells escape from the mitotic arrest, even if the SAC is not satisfied, through a process called adaptation or mitotic slippage. Adaptation weakens the killing properties of antimitotics, ultimately giving rise to resistant cancer cells. We summarize here the mechanisms underlying this process and propose a strategy to identify the factors involved using budding yeast as a model system. Inhibition of factors involved in SAC adaptation could have important therapeutic applications by potentiating the ability of antimitotics to cause cell death.

Zds1/Zds2-PP2ACdc55 complex specifies signaling output from Rho1 GTPase.

Budding yeast Rho1 guanosine triphosphatase (GTPase) plays an essential role in polarized cell growth by regulating cell wall glucan synthesis and actin organization. Upon cell wall damage, Rho1 blocks polarized cell growth and repairs the wounds by activating the cell wall integrity (CWI) Pkc1-mitogen-activated protein kinase (MAPK) pathway. A fundamental question is how active Rho1 promotes distinct signaling outputs under different conditions. Here we identified the Zds1/Zds2-protein phosphatase 2A(Cdc55) (PP2A(Cdc55)) complex as a novel Rho1 effector that regulates Rho1 signaling specificity. Zds1/Zds2-PP2A(Cdc55) promotes polarized growth and cell wall synthesis by inhibiting Rho1 GTPase-activating protein (GAP) Lrg1 but inhibits CWI pathway by stabilizing another Rho1 GAP, Sac7, suggesting that active Rho1 is biased toward cell growth over stress response. Conversely, upon cell wall damage, Pkc1-Mpk1 activity inhibits cortical PP2A(Cdc55), ensuring that Rho1 preferentially activates the CWI pathway for cell wall repair. We propose that PP2A(Cdc55) specifies Rho1 signaling output and that reciprocal antagonism between Rho1-PP2A(Cdc55) and Rho1-Pkc1 explains how only one signaling pathway is robustly activated at a time.

The budding yeast Polo-like kinase Cdc5 is released from the nucleus during anaphase for timely mitotic exit.

Polo-like kinases are important regulators of multiple mitotic events; however, how Polo-like kinases are spatially and temporally regulated to perform their many tasks is not well understood. Here, we examined the subcellular localization of the budding yeast Polo-like kinase Cdc5 using a functional Cdc5-GFP protein expressed from the endogenous locus. In addition to the well-described localization of Cdc5 at the spindle pole bodies (SPBs) and the bud neck, we found that Cdc5-GFP accumulates in the nucleus in early mitosis but is released to the cytoplasm in late mitosis in a manner dependent on the Cdc14 phosphatase. This Cdc5 release from the nucleus is important for mitotic exit because artificial sequestration of Cdc5 in the nucleus by addition of a strong nuclear localization signal (NLS) resulted in mitotic exit defects. We identified a key cytoplasmic target of Cdc5 as Bfa1, an inhibitor of mitotic exit. Our study revealed a novel layer of Cdc5 regulation and suggests the existence of a possible coordination between Cdc5 and Cdc14 activity.

Comparative genetic analysis of PP2A-Cdc55 regulators in budding yeast.

Cdc55, a regulatory B subunit of the protein phosphatase 2A (PP2A) complex, plays various functions during mitosis. Sequestration of Cdc55 from the nucleus by Zds1 and Zds2 is important for robust activation of mitotic Cdk1 and mitotic progression in budding yeast. However, Zds1-family proteins are found only in fungi but not in higher eukaryotes. In animal cells, highly conserved ENSA/ARPP-19 family proteins bind and inhibit PP2A-B55 activity for mitotic entry.   In this study, we compared the relative contribution of Zds1/Zds2 and ENSA-family proteins Igo1/Igo2 on Cdc55 functions in budding yeast mitosis. We confirmed that Igo1/Igo2 can inhibit Cdc55 in early mitosis, but their contribution to Cdc55 regulation is relatively minor compared with the role of Zds1/Zds2. In contrast to Zds1, which primarily localized to the sites of cell polarity and in the cytoplasm, Igo1 is localized in the nucleus, suggesting that Igo1/Igo2 inhibit Cdc55 in a manner distinct from Zds1/Zds2. Our analysis confirmed an evolutionarily conserved function of ENSA-family proteins in inhibiting PP2A-Cdc55, and we propose that Zds1-dependent sequestration of PP2A-Cdc55 from the nucleus is uniquely evolved to facilitate closed mitosis in fungal species.