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SummaryThe proteome provides unique insights into biology and disease beyond the genome and transcriptome. Lack of large proteomic datasets has restricted identification of new cancer biomarkers. Here, proteomes of 949 cancer cell lines across 28 tissue types were analyzed by mass spectrometry. Deploying a clinically-relevant workflow to quantify 8,498 proteins, these data capture evidence of cell type and post-transcriptional modifications. Integrating multi-omics, drug response and CRISPR-Cas9 gene essentiality screens with a deep learning-based pipeline revealed thousands of protein-specific biomarkers of cancer vulnerabilities. Proteomic data had greater power to predict drug response than the equivalent portion of the transcriptome. Further, random downsampling to only 1,500 proteins had limited impact on predictive power, consistent with protein networks being highly connected and co-regulated. This pan-cancer proteomic map (ProCan-DepMapSanger), available at https://cellmodelpassports.sanger.ac.uk, is a comprehensive resource revealing principles of protein regulation with important implications for future clinical studies.

The proteome provides unique insights into disease biology beyond the genome and transcriptome. A lack of large proteomic datasets has restricted the identification of new cancer biomarkers. Here, proteomes of 949 cancer cell lines across 28 tissue types are analyzed by mass spectrometry. Deploying a workflow to quantify 8,498 proteins, these data capture evidence of cell-type and post-transcriptional modifications. Integrating multi-omics, drug response, and CRISPR-Cas9 gene essentiality screens with a deep learning-based pipeline reveals thousands of protein biomarkers of cancer vulnerabilities that are not significant at the transcript level. The power of the proteome to predict drug response is very similar to that of the transcriptome. Further, random downsampling to only 1,500 proteins has limited impact on predictive power, consistent with protein networks being highly connected and co-regulated. This pan-cancer proteomic map (ProCan-DepMapSanger) is a comprehensive resource available at https://cellmodelpassports.sanger.ac.uk.

Reproducible research is the bedrock of experimental science. To enable the deployment of large-scale proteomics, we assess the reproducibility of mass spectrometry (MS) over time and across instruments and develop computational methods for improving quantitative accuracy. We perform 1560 data independent acquisition (DIA)-MS runs of eight samples containing known proportions of ovarian and prostate cancer tissue and yeast, or control HEK293T cells. Replicates are run on six mass spectrometers operating continuously with varying maintenance schedules over four months, interspersed with ~5000 other runs. We utilise negative controls and replicates to remove unwanted variation and enhance biological signal, outperforming existing methods. We also design a method for reducing missing values. Integrating these computational modules into a pipeline (ProNorM), we mitigate variation among instruments over time and accurately predict tissue proportions. We demonstrate how to improve the quantitative analysis of large-scale DIA-MS data, providing a pathway toward clinical proteomics., Nature Communications, 11 (1), ISSN:2041-1723

A standardized procedure for label-free nano-LC-SRM analysis of 32 high-medium abundance proteins from nondepleted human plasma was established and SRM data were acquired on 45 separate days for a control sample that was independently prepared on 39 distinct dates over an 18-month period (542 days). This case study enabled us to assess quantitative variance associated with nano-LC-SRM plasma analysis, mimicking experimental conditions that would be experienced with clinical trial biomarker studies. We assessed sample preparation variability attributed to different technicians and sample storage stability. Instrument performance varied over the 18-month period requiring ion path cleaning, so we assessed the impact of declining performance on specific peptide ion sensitivity and evaluated how various data normalization strategies could compensate for these changes. Our analysis demonstrated that while sample preparation was the main contributor for data variances when MS data were acquired within days, variability in SRM sensitivity was a far greater source of variance when data were acquired over a long period. The overall median multiplexed assay CV was 13% over the 18-month period. This case study is illustrative of large-scale plasma biomarker studies using nano-LC-SRM over extended periods and highlights aspects of bioanalysis that require careful attention to ensure reliable quantitation.

We have developed a streamlined proteomic sample preparation protocol termed Accelerated Barocycler Lysis and Extraction (ABLE) that substantially reduces the time and cost of tissue sample processing. ABLE is based on pressure cycling technology (PCT) for rapid tissue solubilization and reliable, controlled proteolytic digestion. Here, a previously reported PCT based protocol was optimized using 1-4 mg biopsy punches from rat kidney. The tissue denaturant urea was substituted with a combination of sodium deoxycholate (SDC) and N-propanol. ABLE produced comparable numbers of protein identifications in half the sample preparation time, being ready for MS injection in 3 h compared with 6 h for the conventional urea based method. To validate ABLE, it was applied to a diverse range of rat tissues (kidney, lung, muscle, brain, testis), human HEK 293 cell lines, and human ovarian cancer samples, followed by SWATH-mass spectrometry (SWATH-MS). There were similar numbers of quantified proteins between ABLE-SWATH and the conventional method, with greater than 70% overlap for all sample types, except muscle (58%). The ABLE protocol offers a standardized, high-throughput, efficient, and reproducible proteomic preparation method that when coupled with SWATH-MS has the potential to accelerate proteomics analysis to achieve a clinically relevant turn-around time.

Low molecular weight peptides derived from the breakdown of crystallins have been reported in adult human lenses. The proliferation of these LMW peptides coincides with the earliest stages of cataract formation, suggesting that the protein cleavages involved may contribute to the aggregation and insolubilization of crystallins. This study reports the identification of 238 endogenous LMW crystallin peptides from the cortical extracts of four human lenses representing young, middle and old-age human lenses. Analysis of the peptide terminal amino acids showed that Lys and Arg were situated at the C-terminus with significantly higher frequency compared to other residues, suggesting that trypsin-like proteolysis may be active in the lens cortical fiber cells. Selected reaction monitoring analysis of an endogenous αA-crystallin peptide (αA(57-65)) showed that the concentration of this peptide in the human lens increased gradually to middle age, after which the rate of αA(57-65) formation escalated significantly. Using 2D gel electrophoresis/nanoLC-ESI-MS/MS, 12 protein complexes of 40-150 kDa consisting of multiple crystallin components were characterized from the water soluble cortical extracts of an adult human lens. The detection of these protein complexes suggested the possibility of crystallin cross-linking, with these complexes potentially acting to stabilize degraded crystallins by sequestration into water soluble complexes.

The treatment of melanoma by targeted inhibition of the mutated kinase BRAF with small molecules only temporarily suppresses metastatic disease. In the face of chemical inhibition tumor plasticity, both innate and adaptive, promotes survival through the biochemical and genetic reconfiguration of cellular pathways that can engage proliferative and migratory systems. To investigate this process, high-resolution mass spectrometry was used to characterize the phosphoproteome of this transition in vitro. A simple and accurate, label-free quantitative method was used to localize and quantitate thousands of phosphorylation events. We also correlated changes in the phosphoproteome with the proteome to more accurately determine changes in the activity of regulatory kinases determined by kinase landscape profiling. The abundance of phosphopeptides with sites that function in cytoskeletal regulation, GTP/GDP exchange, protein kinase C, IGF signaling, and melanosome maturation were highly divergent after transition to a drug resistant phenotype.

Summary The centre of the human lens, which is composed of proteins that were synthesized prior to birth, is an ideal model for the evaluation of long-term protein stability and processes responsible for the degradation of macromolecules. By analysing the sequences of peptides present in human lens nuclei, characteristic features of intrinsic protein instability were determined. Prominent was the cleavage on the N-terminal side of serine residues. Despite accounting for just 9% of the amino acid composition of crystallins, peptides with N-terminal Ser represented one-quarter of all peptides. Nonenzymatic cleavage at Ser could be reproduced by incubating peptides at elevated temperatures. Serine residues may thus represent susceptible sites for autolysis in polypeptides exposed to physiological conditions over a period of years. Once these sites are cleaved, other chemical processes result in progressive removal or ‘laddering’ of amino acid residues from newly exposed N- and C-termini. As N-terminal Ser peptides originated from several crystallins with unrelated sequences, this may represent a general feature of long-lived proteins.

We performed proteomics analysis on four skin and one muscle tissue samples taken from three ancient Egyptian mummies of the first intermediate period, approximately 4200 years old. The mummies were first dated by radiocarbon dating of the accompany-\break ing textiles, and morphologically examined by scanning electron microscopy of additional skin samples. Proteins were extracted, separated on SDS–PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gels, and in-gel digested with trypsin. The resulting peptides were analysed using nanoflow high-performance liquid chromatography–mass spectrometry. We identified a total of 230 unique proteins from the five samples, which consisted of 132 unique protein identifications. We found a large number of collagens, which was confirmed by our microscopy data, and is in agreement with previous studies showing that collagens are very long-lived. As expected, we also found a large number of keratins. We identified numerous proteins that provide evidence of activation of the innate immunity system in two of the mummies, one of which also contained proteins indicating severe tissue inflammation, possibly indicative of an infection that we can speculate may have been related to the cause of death. This article is part of the themed issue ‘Quantitative mass spectrometry’.

The centre of the human lens, which is composed of proteins that were synthesized prior to birth, is an ideal model for the evaluation of long-term protein stability and processes responsible for the degradation of macromolecules. By analysing the sequences of peptides present in human lens nuclei, characteristic features of intrinsic protein instability were determined. Prominent was the cleavage on the N-terminal side of serine residues. Despite accounting for just 9% of the amino acid composition of crystallins, peptides with N-terminal Ser represented one-quarter of all peptides. Nonenzymatic cleavage at Ser could be reproduced by incubating peptides at elevated temperatures. Serine residues may thus represent susceptible sites for autolysis in polypeptides exposed to physiological conditions over a period of years. Once these sites are cleaved, other chemical processes result in progressive removal or 'laddering' of amino acid residues from newly exposed N- and C-termini. As N-terminal Ser peptides originated from several crystallins with unrelated sequences, this may represent a general feature of long-lived proteins.