Corey E. Bakalarski

ATM and ATR Substrate Analysis Reveals Extensive Protein Networks Responsive to DNA Damage

Cellular responses to DNA damage are mediated by a number of protein kinases, including ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related). The outlines of the signal transduction portion of this pathway are known, but little is known about the physiological scope of the DNA damage response (DDR). We performed a large-scale proteomic analysis of proteins phosphorylated in response to DNA damage on consensus sites recognized by ATM and ATR and identified more than 900 regulated phosphorylation sites encompassing over 700 proteins. Functional analysis of a subset of this data set indicated that this list is highly enriched for proteins involved in the DDR. This set of proteins is highly interconnected, and we identified a large number of protein modules and networks not previously linked to the DDR. This database paints a much broader landscape for the DDR than was previously appreciated and opens new avenues of investigation into the responses to DNA damage in mammals.

Global Survey of Phosphotyrosine Signaling Identifies Oncogenic Kinases in Lung Cancer

Despite the success of tyrosine kinase-based cancer therapeutics, for most solid tumors the tyrosine kinases that drive disease remain unknown, limiting our ability to identify drug targets and predict response. Here we present the first large-scale survey of tyrosine kinase activity in lung cancer. Using a phosphoproteomic approach, we characterize tyrosine kinase signaling across 41 non-small cell lung cancer (NSCLC) cell lines and over 150 NSCLC tumors. Profiles of phosphotyrosine signaling are generated and analyzed to identify known oncogenic kinases such as EGFR and c-Met as well as novel ALK and ROS fusion proteins. Other activated tyrosine kinases such as PDGFRalpha and DDR1 not previously implicated in the genesis of NSCLC are also identified. By focusing on activated cell circuitry, the approach outlined here provides insight into cancer biology not available at the chromosomal and transcriptional levels and can be applied broadly across all human cancers.

A quantitative atlas of mitotic phosphorylation

The eukaryotic cell division cycle is characterized by a sequence of orderly and highly regulated events resulting in the duplication and separation of all cellular material into two newly formed daughter cells. Protein phosphorylation by cyclin-dependent kinases (CDKs) drives this cycle. To gain further insight into how phosphorylation regulates the cell cycle, we sought to identify proteins whose phosphorylation is cell cycle regulated. Using stable isotope labeling along with a two-step strategy for phosphopeptide enrichment and high mass accuracy mass spectrometry, we examined protein phosphorylation in a human cell line arrested in the G1 and mitotic phases of the cell cycle. We report the identification of >14,000 different phosphorylation events, more than half of which, to our knowledge, have not been described in the literature, along with relative quantitative data for the majority of these sites. We observed >1,000 proteins with increased phosphorylation in mitosis including many known cell cycle regulators. The majority of sites on regulated phosphopeptides lie in [S/T]P motifs, the minimum required sequence for CDKs, suggesting that many of the proteins may be CDK substrates. Analysis of non-proline site-containing phosphopeptides identified two unique motifs that suggest there are at least two undiscovered mitotic kinases.

The mitochondrial deubiquitinase USP30 opposes parkin-mediated mitophagy

Cells maintain healthy mitochondria by degrading damaged mitochondria through mitophagy; defective mitophagy is linked to Parkinson’s disease. Here we report that USP30, a deubiquitinase localized to mitochondria, antagonizes mitophagy driven by the ubiquitin ligase parkin (also known as PARK2) and protein kinase PINK1, which are encoded by two genes associated with Parkinson’s disease. Parkin ubiquitinates and tags damaged mitochondria for clearance. Overexpression of USP30 removes ubiquitin attached by parkin onto damaged mitochondria and blocks parkin’s ability to drive mitophagy, whereas reducing USP30 activity enhances mitochondrial degradation in neurons. Global ubiquitination site profiling identified multiple mitochondrial substrates oppositely regulated by parkin and USP30. Knockdown of USP30 rescues the defective mitophagy caused by pathogenic mutations in parkin and improves mitochondrial integrity in parkin- or PINK1-deficient flies. Knockdown of USP30 in dopaminergic neurons protects flies against paraquat toxicity in vivo, ameliorating defects in dopamine levels, motor function and organismal survival. Thus USP30 inhibition is potentially beneficial for Parkinson’s disease by promoting mitochondrial clearance and quality control.

A Proteomic Strategy for Gaining Insights into Protein Sumoylation in Yeast

Sumoylation represents a vital post-translational modification that pervades numerous aspects of cell biology, including protein targeting, transcriptional regulation, signal transduction, and cell division. However, despite its broad reaching effects, most biological outcomes of protein sumoylation remain poorly understood. In an effort to provide further insight into this complex process, a proteomics approach was undertaken to identify the targets of sumoylation en mass. Specifically, SUMO-conjugated proteins were isolated by a double-affinity purification procedure from a Saccharomyces cerevisiae strain engineered to express tagged SUMO. The components of the isolated protein mixture were then identified by subsequent LC-MS/MS analysis using an LTQ FT mass spectrometer. In this manner, 159 candidate sumoylated proteins were identified by two or more peptides. Furthermore, the high accuracy of the instrument, combined with stringent search criteria, enabled the identification of an additional 92 putative candidates by only one peptide. The validity of this proteomics approach was confirmed by performing subsequent Western blot experiments for numerous proteins and determining the actual sumoylation sites for several other substrates. These data combine with recent works to further our understanding of the breadth and impact of protein sumoylation in a diverse array of biological processes.

Optimization and Use of Peptide Mass Measurement Accuracy in Shotgun Proteomics

Mass spectrometers that provide high mass accuracy such as FT-ICR instruments are increasingly used in proteomic studies. Although the importance of accurately determined molecular masses for the identification of biomolecules is generally accepted, its role in the analysis of shotgun proteomic data has not been thoroughly studied. To gain insight into this role, we used a hybrid linear quadrupole ion trap/FT-ICR (LTQ FT) mass spectrometer for LC-MS/MS analysis of a highly complex peptide mixture derived from a fraction of the yeast proteome. We applied three data-dependent MS/MS acquisition methods. The FT-ICR part of the hybrid mass spectrometer was either not exploited, used only for survey MS scans, or also used for acquiring selected ion monitoring scans to optimize mass accuracy. MS/MS data were assigned with the SEQUEST algorithm, and peptide identifications were validated by estimating the number of incorrect assignments using the composite target/decoy database search strategy. We developed a simple mass calibration strategy exploiting polydimethylcyclosiloxane background ions as calibrant ions. This strategy allowed us to substantially improve mass accuracy without reducing the number of MS/MS spectra acquired in an LC-MS/MS run. The benefits of high mass accuracy were greatest for assigning MS/MS spectra with low signal-to-noise ratios and for assigning phosphopeptides. Confident peptide identification rates from these data sets could be doubled by the use of mass accuracy information. It was also shown that improving mass accuracy at a cost to the MS/MS acquisition rate substantially lowered the sensitivity of LC-MS/MS analyses. The use of FT-ICR selected ion monitoring scans to maximize mass accuracy reduced the number of protein identifications by 40%.

Improved Quantitative Mass Spectrometry Methods for Characterizing Complex Ubiquitin Signals

Ubiquitinated substrates can be recruited to macromolecular complexes through interactions between their covalently bound ubiquitin (Ub) signals and Ub receptor proteins. To develop a functional understanding of the Ub system in vivo, methods are needed to determine the composition of Ub signals on individual substrates and in protein mixtures. Mass spectrometry has emerged as an important tool for characterizing the various forms of Ub. In the Ubiquitin-AQUA approach, synthetic isotopically labeled internal standard peptides are used to quantify unbranched peptides and the branched -GG signature peptides generated by trypsin digestion of Ub signals. Here we have built upon existing methods and established a comprehensive platform for the characterization of Ub signals. Digested peptides and isotopically labeled standards are analyzed either by selected reaction monitoring on a QTRAP mass spectrometer or by narrow window extracted ion chromatograms on a high resolution LTQ-Orbitrap. Additional peptides are now monitored to account for the N terminus of ubiquitin, linear polyUb chains, the peptides surrounding K33 and K48, and incomplete digestion products. Using this expanded battery of peptides, the total amount of Ub in a sample can be determined from multiple loci within the protein, minimizing possible confounding effects of complex Ub signals, digestion abnormalities, or use of mutant Ub in experiments. These methods have been useful for the characterization of in vitro, multistage ubiquitination and have now been extended to reactions catalyzed by multiple E2 enzymes. One question arising from in vitro studies is whether individual protein substrates in cells may be modified by multiple forms of polyUb. Here we have taken advantage of recently developed polyubiquitin linkage-specific antibodies recognizing K48- and K63-linked polyUb chains, coupled with these mass spectrometry methods, to further evaluate the abundance of mixed linkage Ub substrates in cultured mammalian cells. By combining these two powerful tools, we show that polyubiquitinated substrates purified from cells can be modified by mixtures of K48, K63, and K11 linkages.

Complementary Proteomic Tools for the Dissection of Apoptotic Proteolysis Events

Proteolysis is a key regulatory event that controls intracellular and extracellular signaling through irreversible changes in a proteins structure that greatly alters its function. Here we describe a platform for profiling caspase substrates which encompasses two highly complementary proteomic techniques--the first is a differential gel based approach termed Global Analyzer of SILAC-derived Substrates of Proteolysis (GASSP) and the second involves affinity enrichment of peptides containing a C-terminal aspartic acid residue. In combination, these techniques have enabled the profiling of a large cellular pool of apoptotic-mediated proteolytic events across a wide dynamic range. By applying this integrated proteomic work flow to analyze proteolytic events resulting from the induction of intrinsic apoptosis in Jurkat cells via etoposide treatment, 3346 proteins were quantified, of which 360 proteins were identified as etoposide-induced proteolytic substrates, including 160 previously assigned caspase substrates. In addition to global profiling, a targeted approach using BAX HCT116 isogenic cell lines was utilized to dissect pre- and post-mitochondrial extrinsic apoptotic cleavage events. By employing apoptotic activation with a pro-apoptotic receptor agonist (PARA), a limited set of apoptotic substrates including known caspase substrates such as BH3 interacting-domain death agonist (BID) and Poly (ADP-ribose) polymerase (PARP)-1, and novel substrates such as Basic Transcription Factor 3, TRK-fused gene protein (TFG), and p62/Sequestosome were also identified.

Rapid, semi-automated protein terminal characterization using ISDetect

811 To determine an appropriate scoring threshold for this study, we conducted a false positive rate (FPR) analysis to assess the likelihood of random spectral matches to the given protein sequence (Supplementary Fig. 1). Scores of ≥100 were considered unlikely to have arisen by random chance (FPR < 10-5). The software is freely available for download (http://research-pub.gene.com/ proteomics_data/isdetect). We tested ISDetect against 1,787 recombinant proteins across 2,991 analyses using a 2,5-diaminonaphthalene matrix (Supplementary Fig. 2) that would have otherwise undergone Edman degradation (Fig. 2a and Supplementary Fig. 3). ISDetect yielded 5,916 high-confidence results (ISDetect score ≥ 100), identifying at least one terminus for 1,593 (89%) of all proteins assessed (Supplementary Table 1). This included 876 C-terminal sequences that were undetectable with Edman chemistries; furthermore, 34% of proteins (460 of 1,595) were shown to have N-terminal blocking groups, such as acetylation or formylation, and thus were not amenable to N-terminal chemical degradation by Edman methods. In 73% of the remaining non-identifications, follow-up verification showed that the proteins examined were from different sequences or at too low of a concentration for efficient analysis (<0.3 mg ml–1). ISDetect was much faster than automated Edman degradation using fast cycles6 (5 min versus 6 h for 12 cycles). We also confirmed ISDetect results through a comparison to a sample of proteins subjected to Edman analysis, which showed agreement in N-terminal identification in all ten proteins assessed (Supplementary Table 2). Furthermore, ISDetect was also able to verify all protein termini in a three-protein mixture (Supplementary Table 3). We also compared our results to those obtained through pseudo-MS/MS analysis using Mascot. Although Mascot was originally developed for peptide-level proteomic analysis, it has been used for ISD spectral interpretation as a part of vendor software used in recent studies7. Of 250 protein samples randomly selected from high-scoring ISDetect results, Mascot validated at least one terminus in 36.4% cases (91 of 250 peptide fragments; Supplementary Table 4). In contrast to Edman degradation, ISDetect automatically detects and determines the mass of post-translational modifications at either terminus. Reports suggest that up to 80% of proteins are To the Editor: Terminal characterization of purified proteins is a powerful and widely used analytical tool for verification of recombinant protein identity, assessment of signal sequence removal for X-ray crystallography1, characterization of intracellular proteolysis events, and confirmation of protein de novo sequencing2. The chemistries described by Pehr Edman3 for N-terminal sequence characterization over 50 years ago remain the gold-standard approach for identifying protein termini. Although Edman degradation is well validated, limitations in coverage, speed, and versatility have prompted a search for alternative methods. Matrix-assisted laser desorption/ ionization–in-source decay (MALDIISD) has previously been used to confirm protein identity, for therapeutic antibody verification, and as an aid in tissue imaging. In MALDI-ISD, purified protein is combined with a proton-donating matrix reagent on a MALDI target plate. Upon laser ablation, fragmentation occurs at either protein terminus, resulting in the creation of fragment ions that can be detected by a mass analyzer and exploited to yield partial primary sequence information4. Widespread implementation of MALDI-ISD has been hampered by the lack of broadly applicable tools to ease the burden of manual interpretation, which can take hours per spectrum. Here we report the development of ISDetect, a software tool that uses mass spectrometry for rapid protein terminal sequence verification. ISDetect uses a probability-based approach for rapid, automated matching of ISD spectra against a putative sequence or a short list of candidate sequences. The algorithm provides sequence matches regardless of cleavage events, spectral complexity, or terminal modifications, and it can define both N and C termini simultaneously from a single spectrum. It can be applied to data derived from any MALDI–timeof-flight (TOF/TOF) mass spectrometer. Additionally, it automatically detects cleavage events and post-translational terminal modifications, allowing identification of clipped and ragged termini, a unique feature among existing methods of MALDI-ISD analysis. Most important, automation alleviates the laborious process of manual interpretation, reducing analysis times from hours to minutes and enabling use for highthroughput validation. The ISDetect algorithm (Fig. 1) accepts a list of m/z and intensity values from a MALDI-ISD spectrum and matches it against a supplied protein sequence. After a noisereducing pruning step, the algorithm searches for peak pair tags with mass differences matching amino acids contained in the putative sequence. Matches are extended to identify additional peak pairs consistent with the putative sequence. Peaks may be contiguous or noncontiguous; gaps in the fragment sequence are ignored. Partial sequence matches are then scored by a binomial spectral peak model to rank the likelihood of the matches observed; similar models have proven effective in the related fields of tandem MS/MS spectral interpretation5 and post-translational modification site localization. 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