Proteome Extraction Research Articles

Differential Expression Analysis of Cutaneous Squamous Cell Carcinoma and Basal Cell Carcinoma Proteomic Profiles Sampled with Electroporation-Based Biopsy

Clinical misdiagnosis between cutaneous squamous cell carcinoma (cSCC) and basal cell carcinoma (BCC) affects treatment plans. We report a tissue sampling approach with molecular biopsy using electroporation. This method, coined e-biopsy, enables non-destructive non-thermal permeabilization of cells in the skin for vacuum-assisted extraction of biomolecules. We used e-biopsy for ex vivo proteome extraction from 3 locations per patient in 21 cSCC, 20 BCC and 7 actinic keratosis human skin samples. Using liquid chromatography mass spectrometry (LC/MS/MS), we identified 5,966 proteins observed with non-zero intensity in at least one samples. The intra-patient Pearson correlation of 0.888 ± 0.065 for BCC patients; 0.858 ± 0.077 for SCC patients; 0.876 ± 0.116 for solar actinic keratosis (AK) patients indicates high consistency of the e-biopsy sampling. The mass spectra presented significantly different proteome profiles for cSCC, BCC and AK, with several hundreds of proteins differentially expressed. Notably, our study showed that proteomes sampled with e-biopsy from cSCC and BCC lesions are different, and that proteins of CRNN, SULT1E1 and ITPK1 genes are significantly overexpressed in BCC in comparison to cSCC. Our results provide evidence that the e-biopsy approach could potentially be used as a tool to support cutaneous lesions classification with molecular pathology.

Impact of Surfactants on Cumulative Trypsin Activity in Bottom-Up Proteome Analysis.

Trypsin digestion plays a pivotal role in successful bottom-up peptide characterization and quantitation. While denaturants are often incorporated to enhance protein solubility, surfactants are recognized to inhibit enzyme activity. However, several reports have suggested that incorporating surfactants or other solvent additives may enhance digestion and MS detection. Here, we assess the impacts of ionic surfactants on cumulative trypsin activity and subsequently evaluate the total digestion efficiency of a proteome mixture by quantitative MS. Although low surfactant concentrations, such as 0.01% SDS or 0.2% SDC, significantly enhanced the initial trypsin activity (by 14 or 42%, respectively), time course assays revealed accelerated enzyme deactivation, evident by 10- or 40-fold reductions in trypsin activity half-life at these respective surfactant concentrations. Despite enhanced initial tryptic activity, quantitative MS analysis of a common liver proteome extract, digested with various surfactants (0.01 or 0.1% SDS, 0.5% SDC), consistently revealed decreased peptide counts and signal intensity, indicative of a lower digestion efficiency compared to a nonsurfactant control. Furthermore, including detergents for digestion did not improve the detection of membrane proteins, nor hydrophobic peptides. These results stress the importance of assessing cumulative enzyme activity when optimizing the digestion of a proteome mixture, particularly in the presence of denaturants.

Deciphering dermatological distinctions: Cornulin as a discriminant biomarker between basal cell carcinoma and squamous cell carcinoma detected through e‐biopsy and machine learning

AbstractClinical misdiagnosis between cutaneous squamous cell carcinoma (cSCC) and basal cell carcinoma (BCC) poses treatment challenges and carries risks of recurrence, metastases and increased morbidity and mortality. We aimed to identify discriminant proteins markers for cSCC and BCC using a minimally invasive proteome sampling method called e‐biopsy, employing electroporation for non‐thermal cell permeabilization and machine learning. E‐biopsy facilitated ex vivo proteome extraction from 21 cSCC and 21 BCC pathologically validated human cancers. LC/MS/MS profiling of 126 proteomes was followed by machine learning analysis to identify proteins distinguishing cSCC from BCC. For identified panel validation, we used proteomes sampled by e‐biopsy from unrelated 20 cSCC and 46 BCC human cancers, and differential expression analysis of published transcriptomics. Cornulin, the most commonly chosen discriminative biomarker by machine learning models, was also validated using fluorescent immunohistochemistry. One hundred and ninety‐two proteomes sampled from one hundred eight patients were analysed. Machine learning‐based approaches resulted in a set of 11 potential biomarker proteins that can be used to construct a patient classification model with 95.2% average cross‐validation accuracy, BCC precision of 93.6 ± 14.5%, cSCC precision of 98.4 ± 7.2%, specificity of 97.7 ± 11.8% and sensitivity 92.7 ± 15.3%. Protein–protein interaction analysis revealed a novel interaction network connecting 10 of the 11 resulted proteins. Histological and transcriptomic validation confirmed cornulin as a discriminant marker significantly lower in cSCC than in BCC. E‐biopsy combined with machine learning provides a novel approach to molecular sampling from skin for biomarker detection and differential expression analysis between cSCC and BCC.

Comparing extraction method efficiency for high-throughput palaeoproteomic bone species identification

High-throughput proteomic analysis of archaeological skeletal remains provides information about past fauna community compositions and species dispersals in time and space. Archaeological skeletal remains are a finite resource, however, and therefore it becomes relevant to optimize methods of skeletal proteome extraction. Ancient proteins in bone specimens can be highly degraded and consequently, extraction methods for well-preserved or modern bone might be unsuitable for the processing of highly degraded skeletal proteomes. In this study, we compared six proteomic extraction methods on Late Pleistocene remains with variable levels of proteome preservation. We tested the accuracy of species identification, protein sequence coverage, deamidation, and the number of post-translational modifications per method. We find striking differences in obtained proteome complexity and sequence coverage, highlighting that simple acid-insoluble proteome extraction methods perform better in highly degraded contexts. For well-preserved specimens, the approach using EDTA demineralization and protease-mix proteolysis yielded a higher number of identified peptides. The protocols presented here allowed protein extraction from ancient bone with a minimum number of working steps and equipment and yielded protein extracts within three working days. We expect further development along this route to benefit large-scale screening applications of relevance to archaeological and human evolution research.

Extracting proteins from microscopic biominerals: A reproducible method development using oyster larvae

AbstractMicroscopic biominerals are ubiquitous in the ocean, and several major taxa secrete them during early life stages or as adults. Organisms secrete an extracellular proteome incorporated within the biomineral to guide biomineralization remotely and enhance its material properties. This proteome has attracted the attention of extensive scientific research, but its characterization is challenging due to methodological constraints that limit the overall insight, particularly in small organisms. Therefore, we propose this straightforward and reproducible method development for preparing microscopic biominerals before proteome extraction. The method development can be tailored to other microscopic biominerals, and, importantly, it aims to integrate biomineral cleanliness and integrity without sacrificing proteome completeness. First, we suggest running an in‐depth sample exploration to identify key sample characteristics and determine the magnitude of the sodium hypochlorite (NaOCl) treatment. Then, we recommend running a multiple time points experiment for biomineral cleaning treatment with a fixed NaOCl concentration. The time points are evaluated using qualitative (visual assessment) and quantitative methods (biomineral loss, elemental composition, and organic structural components removal). Finally, critical time points are identified for method validation using shotgun proteomics. This approach was tested using Hong Kong oyster larval shells as a model organism. Our study discovered that surprisingly, longer treatments and partial biomineral damage are preferred for Hong Kong oyster larvae and do not lead to protein diversity loss but enrichment. This microscopic biomineral cleaning method development can facilitate harnessing information from increasingly diverse biomineral proteomes.

Optimized Proteome Reduction for Integrative Top-Down Proteomics.

Integrative top-down proteomics is an analytical approach that fully addresses the breadth and complexity needed for effective and routine assessment of proteomes. Nonetheless, any such assessments also require a rigorous review of methodology to ensure the deepest possible quantitative proteome analyses. Here, we establish an optimized general protocol for proteome extracts to improve the reduction of proteoforms and, thus, resolution in 2DE. Dithiothreitol (DTT), tributylphosphine (TBP), and 2-hydroxyethyldisulfide (HED), combined and alone, were tested in one-dimensional SDS-PAGE (1DE), prior to implementation into a full 2DE protocol. Prior to sample rehydration, reduction with 100 mM DTT + 5 mM TBP yielded increased spot counts, total signal, and spot circularity (i.e., decreased streaking) compared to other conditions and reduction protocols reported in the literature. The data indicate that many widely implemented reduction protocols are significantly 'under-powered' in terms of proteoform reduction and thus, limit the quality and depth of routine top-down proteomic analyses.

Proteome Coverage after Simultaneous Proteo-Metabolome Liquid-Liquid Extraction.

Proteomics and metabolomics are essential in systems biology, and simultaneous proteo-metabolome liquid-liquid extraction (SPM-LLE) allows isolation of the metabolome and proteome from the same sample. Since the proteome is present as a pellet in SPM-LLE, it must be solubilized for quantitative proteomics. Solubilization and proteome extraction are critical factors in the information obtained at the proteome level. In this study, we investigated the performance of two surfactants (sodium deoxycholate (SDC), sodium dodecyl sulfate (SDS)) and urea in terms of proteome coverage and extraction efficiency of an interphase proteome pellet generated by methanol-chloroform based SPM-LLE. We also investigated how the performance differs when the proteome is extracted from the interphase pellet or by direct cell lysis. We quantified 12 lipids covering triglycerides and various phospholipid classes, and 25 polar metabolites covering central energy metabolism in chloroform and methanol extracts. Our study reveals that the proteome coverages between the two surfactants and urea for the SPM-LLE interphase pellet were similar, but the extraction efficiencies differed significantly. While SDS led to enrichment of basic proteins, which were mainly ribosomal and ribonuclear proteins, urea was the most efficient extraction agent for simultaneous proteo-metabolome analysis. The results of our study also show that the performance of surfactants for quantitative proteomics is better when the proteome is extracted through direct cell lysis rather than an interphase pellet. In contrast, the performance of urea for quantitative proteomics was significantly better when the proteome was extracted from an interphase pellet than by direct cell lysis. We demonstrated that urea is superior to surfactants for proteome extraction from SPM-LLE interphase pellets, with a particularly good performance for the extraction of proteins associated with metabolic pathways. Data are available via ProteomeXchange with identifier PXD027338.

ASAP─Automated Sonication-Free Acid-Assisted Proteomes─from Cells and FFPE Tissues.

Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable resource for retrospective studies, but protein extraction and subsequent sample processing steps have been shown to be challenging for mass spectrometry (MS) analysis. Streamlined high-throughput sample preparation workflows are essential for efficient peptide extraction from complex clinical specimens such as fresh frozen tissues or FFPE. Overall, proteome analysis has gained significant improvements in the instrumentation, acquisition methods, sample preparation workflows, and analysis pipelines, yet even the most recent FFPE workflows remain complex and are not readily scalable. Here, we present an optimized workflow for automated sonication-free acid-assisted proteome (ASAP) extraction from FFPE sections. ASAP enables efficient protein extraction from FFPE specimens, achieving similar proteome coverage as established methods using expensive sonicators, resulting in reduced sample processing time. The broad applicability of ASAP on archived pediatric tumor FFPE specimens resulted in high-quality data with increased proteome coverage and quantitative reproducibility. Our study demonstrates the practicality and superiority of the ASAP workflow as a streamlined, time- and cost-effective pipeline for high-throughput FFPE proteomics of clinical specimens.

Evaluation of Two Simultaneous Metabolomic and Proteomic Extraction Protocols Assessed by Ultra-High-Performance Liquid Chromatography Tandem Mass Spectrometry.

Untargeted multi-omics analysis of plasma is an emerging tool for the identification of novel biomarkers for evaluating disease prognosis, and for developing a better understanding of molecular mechanisms underlying human disease. The successful application of metabolomic and proteomic approaches relies on reproducibly quantifying a wide range of metabolites and proteins. Herein, we report the results of untargeted metabolomic and proteomic analyses from blood plasma samples following analyte extraction by two frequently-used solvent systems: chloroform/methanol and methanol-only. Whole blood samples were collected from participants (n = 6) at University Hospital Sharjah (UHS) hospital, then plasma was separated and extracted by two methods: (i) methanol precipitation and (ii) 4:3 methanol:chloroform extraction. The coverage and reproducibility of the two methods were assessed by ultra-high-performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-ESI-QTOF-MS). The study revealed that metabolite extraction by methanol-only showed greater reproducibility for both metabolomic and proteomic quantifications than did methanol/chloroform, while yielding similar peptide coverage. However, coverage of extracted metabolites was higher with the methanol/chloroform precipitation.

An autoantigen profile from Jurkat T-Lymphoblasts provides a molecular guide for investigating autoimmune sequelae of COVID-19

In order to understand autoimmune phenomena contributing to the pathophysiology of COVID-19 and post-COVID syndrome, we have been profiling autoantigens (autoAgs) from various cell types. Although cells share numerous autoAgs, each cell type gives rise to unique COVID-altered autoAg candidates, which may explain the wide range of symptoms experienced by patients with autoimmune sequelae of SARS-CoV-2 infection. Based on the unifying property of affinity between autoAgs and the glycosaminoglycan dermatan sulfate (DS), this paper reports 140 candidate autoAgs identified from proteome extracts of human Jurkat T-cells, of which at least 105 (75%) are known targets of autoantibodies. Comparison with currently available multi-omic COVID-19 data shows that 125 (89%) DS-affinity proteins are altered at protein and/or RNA levels in SARS-CoV-2-infected cells or patients, with at least 94 being known autoAgs in a wide spectrum of autoimmune diseases and cancer. Protein alterations by ubiquitination and phosphorylation during the viral infection are major contributors of autoAgs. The autoAg protein network is significantly associated with cellular response to stress, apoptosis, RNA metabolism, mRNA processing and translation, protein folding and processing, chromosome organization, cell cycle, and muscle contraction. The autoAgs include clusters of histones, CCT/TriC chaperonin, DNA replication licensing factors, proteasome and ribosome proteins, heat shock proteins, serine/arginine-rich splicing factors, 14-3-3 proteins, and cytoskeletal proteins. AutoAgs, such as LCP1 and NACA, that are altered in the T cells of COVID patients may provide insight into T-cell responses to viral infection and merit further study. The autoantigen-ome from this study contributes to a comprehensive molecular map for investigating acute, subacute, and chronic autoimmune disorders caused by SARS-CoV-2.

In situ cell-type-specific cell-surface proteomic profiling in mice

Cell-surface proteins (CSPs) mediate intercellular communication throughout the lives of multicellular organisms. However, there are no generalizable methods for quantitative CSP profiling in specific cell types in vertebrate tissues. Here, we present in situ cell-surface proteome extraction by extracellular labeling (iPEEL), a proximity labeling method in mice that enables spatiotemporally precise labeling of cell-surface proteomes in a cell-type-specific environment in native tissues for discovery proteomics. Applying iPEEL to developing and mature cerebellar Purkinje cells revealed differential enrichment in CSPs with post-translational protein processing and synaptic functions in the developing and mature cell-surface proteomes, respectively. A proteome-instructed invivo loss-of-function screen identified a critical, multifaceted role for Armh4 in Purkinje cell dendrite morphogenesis. Armh4 overexpression also disrupts dendrite morphogenesis; this effect requires its conserved cytoplasmic domain and is augmented by disrupting its endocytosis. Our results highlight the utility of CSP profiling in native mammalian tissues for identifying regulators of cell-surface signaling.

Maximizing Cumulative Trypsin Activity with Calcium at Elevated Temperature for Enhanced Bottom-Up Proteome Analysis.

Simple SummaryTrypsin is frequently employed to cleave proteins ahead of mass spectrometry characterization. Traditionally, enzyme digestion involves overnight incubation of proteins at 37 °C, which is time consuming though still may yield poor digestion efficiency. While raising the temperature should theoretically accelerate the digestion, it also destabilizes the enzyme and promotes trypsin de-activation. We therefore questioned whether elevated temperature is beneficial for improving tryptic digestion. Here, we quantify protein digestion kinetics at elevated temperatures for calcium-stabilized trypsin and enforce the critical importance of calcium ions to preserve the enzyme. We quantitatively demonstrate that 1 h at 47 °C provides a superior digest when compared to conventional (overnight, 37 °C) processing of the proteome. The practical impact of our enhanced digestion protocol is shown through bottom-up mass spectrometry analysis of a complex proteome mixture.Bottom-up proteomics relies on efficient trypsin digestion ahead of MS analysis. Prior studies have suggested digestion at elevated temperature to accelerate proteolysis, showing an increase in the number of MS-identified peptides. However, improved sequence coverage may be a consequence of partial digestion, as higher temperatures destabilize and degrade the enzyme, causing enhanced activity to be short-lived. Here, we use a spectroscopic (BAEE) assay to quantify calcium-stabilized trypsin activity over the complete time course of a digestion. At 47 °C, the addition of calcium contributes a 25-fold enhancement in trypsin stability. Higher temperatures show a net decrease in cumulative trypsin activity. Through bottom-up MS analysis of a yeast proteome extract, we demonstrate that a 1 h digestion at 47 °C with 10 mM Ca2+ provides a 29% increase in the total number of peptide identifications. Simultaneously, the quantitative proportion of peptides with 1 or more missed cleavage sites was diminished in the 47 °C digestion, supporting enhanced digestion efficiency with the 1 h protocol. Trypsin specificity also improves, as seen by a drop in the quantitative abundance of semi-tryptic peptides. Our enhanced digestion protocol improves throughput for bottom-up sample preparation and validates the approach as a robust, low-cost alternative to maximized protein digestion efficiency.

The effect of eraser sampling for proteomic analysis on Palaeolithic bone surface microtopography

Bone surface modifications are crucial for understanding human subsistence and dietary behaviour, and can inform about the techniques employed in the production and use of bone tools. Permission to destructively sample such unique artefacts is not always granted. The recent development of non-destructive proteomic extraction techniques has provided some alternatives for the analysis of rare and culturally significant artefacts, including bone tools and personal ornaments. The Eraser Extraction Method (EEM), first developed for ZooMS analysis of parchment, has recently been applied to bone and ivory specimens. To test the potential impact of the EEM on ancient bone surfaces, we analyse six anthropogenically modified Palaeolithic bone specimens from Bacho Kiro Cave (Bulgaria) through a controlled sampling experiment using qualitative and 3D quantitative microscopy. Although the overall bone topography is generally preserved, our findings demonstrate a slight flattening of the microtopography alongside the formation of micro-striations associated with the use of the eraser for all bone specimens. Such modifications are similar to ancient use-wear traces. We therefore consider the EEM a destructive sampling approach for Palaeolithic bone surfaces. Together with low ZooMS success rates in some of the reported studies, the EEM might not be a suitable approach to taxonomically identify Pleistocene bone specimens.

Salt-Mediated Organic Solvent Precipitation for Enhanced Recovery of Peptides Generated by Pepsin Digestion.

Conventional solvent-based precipitation makes it challenging to obtain a high recovery of low mass peptides. However, we previously demonstrated that the inclusion of salt ions, specifically ZnSO4, together with high concentrations of acetone, maximizes the recovery of peptides generated from trypsin digestion. We herein generalized this protocol to the rapid (5 min) precipitation of pepsin-digested peptides recovered from acidic matrices. The precipitation protocol extended to other organic solvents (acetonitrile), with high recovery from dilute peptide samples permitting preconcentration and purification. Mass spectrometry profiling of pepsin-generated peptides demonstrated that the protocol captured peptides as small as 800 u, although with a preferential bias towards recovering larger and more hydrophobic peptides. The precipitation protocol was applied to rapidly quench, concentrate, and purify pepsin-digested samples ahead of MS. Complex mixtures of yeast and plasma proteome extracts were successfully precipitated following digestion, with over 95% of MS-identified peptides observed in the pellet fraction. The full precipitation workflow—including the digestion step—can be completed in under 10 min, with direct MS analysis of the recovered peptide pellets showing exceptional protein sequence coverage.

Automated Electrokinetic Platform for High-Throughput Sodium Dodecyl Sulfate Depletion Ahead of Proteome Analysis by Mass Spectrometry.

Sodium dodecyl sulfate (SDS) provides numerous benefits for proteome sample preparation. However, the surfactant can be detrimental to downstream mass spectrometry analysis. Although strategies are available to deplete SDS from proteins, each is plagued by unique deficiencies that challenge their utility for high-throughput proteomics. An optimal approach would rapidly and reproducibly achieve less than 10 ppm residual SDS while simultaneously maximizing analyte recovery. Here, we describe improvements to a simple electrokinetic device termed transmembrane electrophoresis, which we previously reported for automated, rapid SDS depletion of proteome samples. Voltage-driven transport of SDS across a molecular weight cutoff membrane is enhanced at higher electric fields, which is herein achieved by integrating an active cooling mechanism to mitigate the impacts of Joule heating. We report 99.9% reduction of SDS (final concentration < 5 ppm) in 5 min. The device is employed in a detergent-based proteomic workflow for analysis of an enriched yeast membrane proteome extract, demonstrating quantitative protein recovery (>98%) and increasing the number of identifications by liquid chromatography-tandem mass spectrometry.

Advances of ionic liquids-based novel methods for proteomic sample preparation

<p indent=0mm> In-depth coverage analysis of proteome can detect the dynamic changes of protein expression in a panoramic view, which is of great significance for revealing the mechanisms of disease occurrence and development to discover new drug targets. Sample preparation is the prerequisite for proteomic analysis, in which protein extraction is crucial and affects the coverage of qualitative and quantitative analysis for proteome. However, the existing extraction reagents have insufficient dissolution ability for hydrophobic proteins, resulting in serious losses during sample preparation. Therefore, it is urgent to develop extraction reagents with strong dissolution ability for proteins, especially for hydrophobic proteins, and at the same time with good compatibility to subsequent enzymatic hydrolysis and liquid chromatography-mass spectrometry analysis. Recently, due to the strong dissolution ability, ionic liquids (ILs) have gradually been used for dissolving, extracting, and separating individual proteins. ILs have also shown their advantages in the preparation of complex proteomic samples, demonstrating excellent proteome extraction performance. Herein, we systematically summarize the IL-based sample preparation methods for membrane proteome, whole proteome and post-translational modification proteome and further explore the interaction mechanism between ILs and proteins. The wide application of ILs in proteomic analysis is also prospected in this review.

Protocol Optimization of Proteomic Analysis of Korean Ginseng (Panax ginseng Meyer)

The benefits of ginseng have been mainly attributed to its triterpenoids, called ginsenosides. Recent genome sequencing of the Panax ginseng has paved the way for in-depth proteomic studies of this medicinal plant. The current study was conducted to deepen the proteomic information on the root proteome of Korean ginseng. Proteomic workflow was optimized by testing two different strategies, characterized by the phenol extraction procedure, the presence or the absence of SDS-PAGE fractionation step, and nano-scale liquid chromatographic tandem mass spectrometry (nLC-MS/MS) analysis. The results highlighted an evident improvement of proteome extraction by the combination of phenol extraction with SDS-PAGE before the nLC-MS/MS analysis. In addition, a dramatic impact of the steaming process (the treatment to produce red ginseng from ginseng) on protein properties was observed. Overall, the analyses of Korean ginseng permitted the characterization of a total of 2412 proteins. A large number of identified proteins belonged to the functional categories of protein and carbon/energy metabolism (22.4% and 14.6%, respectively). The primary and secondary metabolisms are major metabolic pathways, which emerged from the proteomic analysis. In addition, a large number of proteins known to play an important role in response to (a)biotic stresses were also identified. The current proteomic study not only confirmed the previous transcriptomic and proteomic reports but also extended proteomic information, including the main metabolic pathways involved in Korean ginseng.

Proteomic Expression Profile in Human Temporomandibular Joint Dysfunction.

Temporomandibular joint dysfunction (TMD) is a multifactorial condition that impairs human’s health and quality of life. Its etiology is still a challenge due to its complex development and the great number of different conditions it comprises. One of the most common forms of TMD is anterior disc displacement without reduction (DDWoR) and other TMDs with distinct origins are condylar hyperplasia (CH) and mandibular dislocation (MD). Thus, the aim of this study is to identify the protein expression profile of synovial fluid and the temporomandibular joint disc of patients diagnosed with DDWoR, CH and MD. Synovial fluid and a fraction of the temporomandibular joint disc were collected from nine patients diagnosed with DDWoR (n = 3), CH (n = 4) and MD (n = 2). Samples were subjected to label-free nLC-MS/MS for proteomic data extraction, and then bioinformatics analysis were conducted for protein identification and functional annotation. The three TMD conditions showed different protein expression profiles, and novel proteins were identified in both synovial fluid and disc sample. TMD is a complex condition and the identification of the proteins expressed in the three different types of TMD may contribute to a better comprehension of how each pathology develops and evolutes, benefitting the patient with a focus–target treatment.

Novel Proteome Extraction Method Illustrates a Conserved Immunological Signature of MSI-H Colorectal Tumors

Using a simple, environment friendly proteome extraction (TOP), we were able to optimize the analysis of clinical samples. Using our TOP method we analyzed a clinical cohort of microsatellite stable (MSS) and unstable (MSI-H) colorectal carcinoma (CRC). We identified a tumor cell specific, STAT1-centered, immune signature expressed by the MSI-H tumor cells. We then showed that long, but not short, exposure to Interferon-γ induces a similar signature in vitro We identified 10 different temporal protein expression patterns, classifying the Interferon-γ protein temporal regulation in CRC. Our data sheds light on the changes that tumor cells undergo under long-term immunological pressure in vivo, the importance of STAT proteins in specific biological scenarios. The data generated could help find novel clinical biomarkers and therapeutic approaches.

Rapid and Quantitative Protein Precipitation for Proteome Analysis by Mass Spectrometry.

Protein precipitation is a common front-end preparation strategy for proteome analysis, as well as other applications (e.g., protein depletion for small molecule analysis, bulk commercial preparation of protein). Highly variable conditions used to precipitate proteins, ranging in solvent type, strength, time, and temperature, reflect inconsistent and low recovery. As a consequence, incomplete proteome coverage diminishes the utility of precipitation for proteome sample preparation ahead of mass spectrometry. We herein investigate and optimize the conditions affecting protein recovery through precipitation using acetone at a defined ionic strength. By increasing the salt concentration and incubation temperature with 80% acetone, we show that rapid (2 min) precipitation provides consistently high protein recovery (98 ± 1%) of complex proteome extracts. Rapid precipitation is also applicable to isolate dilute proteins starting as low as 1 μg mL-1. Furthermore, analysis of the protein pellet by bottom-up mass spectrometry (MS) reveals unbiased recovery of all proteins with respect to molecular weight, isoelectric point (pI), and hydrophobicity. Our robust strategy to isolate proteins maximizes recovery and throughput, exploiting the analytical advantages of precipitation over alternative techniques. Data are available via ProteomeXchange with identifier PXD015674.