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Journal of proteome research
American Chemical Society
Journal of proteome research

American Chemical Society

1535-3893

Journal of proteome research/Journal Journal of proteome researchISTPSCI
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    Protein Adsorption Loss-The Bottleneck of Single-Cell Proteomics

    Sun, BingyunKumar, Sharwan
    8页
    查看更多>>摘要：Single-cell proteomics is a promising field to provide direct yet comprehensive molecular insights into cellular functions without averaging effects. Here, we address a grand technical challenge impeding the maturation of single-cell proteomics-protein adsorption loss (PAL). Even though widely known, there is currently no quantitation on how profoundly and selectively PAL has affected single-cell proteomics. Therefore, the mitigations to this challenge have been generic, and their efficacy was only evaluated by the size of the resolved proteome with no specificity on individual proteins. We use the existing knowledge of PAL, protein expression, and the typical surface area used in single-cell proteomics to discuss the severity of protein loss. We also summarize the current solutions to this challenge and briefly review the available methods to characterize the physical and chemical properties of protein surface adsorption. By citing successful strategies in single-cell genomics for measurement errors in individual transcripts, we pinpoint the urgency to benchmark PAL at the proteome scale with individual protein resolution. Finally, orthogonal single-cell proteomic techniques that have the potential to cross validate PAL are proposed. We hope these efforts can promote the fruition of single-cell proteomics in the near future.
      原文链接:
    • NSTL
    • Amer Chemical Soc

    Evaluation of Volumetric Absorptive Microsampling and Mass Spectrometry Data-Independent Acquisition of Hemoglobin-Related Clinical Markers

    Lima, Debora A. .Schuch, Rodrigo A. .Salgueiro, Jessica S.Pintao, Maria Carolina T....
    13页
    查看更多>>摘要：Data-independent acquisition (DIA) allows comprehensive proteome coverage, while it also potentially works as a unified protocol to determine a multitude of proteins found in blood . Because of its high specificity, mass spectrometry may greatly reduce the interference observed in other assays to evaluate blood markers. Here, we combined DIA with volumetric absorptive microsampling (VAMS) and automated proteomics sample processing in a platform to assess clinical markers. As a proof of concept, we evaluated two hemoglobin-related biomarkers: the glycated hemoglobin (HbA1c) and hemoglobin (Hb) variants. HbA1c by DIA showed good correlation with the reference method, but method imprecision did not meet the quality requirement for this biomarker. We developed a strategy to identify Hb variants based on a customized database combined with a workflow for DIA data extraction and rigorous peptide evaluation. Data are available via ProteomeXchange with identifier PXD029918.
      原文链接:
    • NSTL
    • Amer Chemical Soc

    SimPLIT: Simplified Sample Preparation for Large-Scale Isobaric Tagging Proteomics

    Sialana, Fernando J.Roumeliotis, Theodoros I.Bouguenina, HabibHak, Laura Chan Wah...
    15页
    查看更多>>摘要：Large scale proteomic profiling of cell lines can reveal molecular signatures attributed to variable genotypes or induced perturbations, enabling proteogenomic associations and elucidation of pharmacological mechanisms of action. Although isobaric labeling has increased the throughput of proteomic analysis, the commonly used sample preparation workflows often require time-consuming steps and costly consumables, limiting their suitability for large scale studies. Here, we present a simplified and cost-effective one-pot reaction workflow in a 96-well plate format (SimPLIT) that minimizes processing steps and demonstrates improved reproducibility compared to alternative approaches. The workflow is based on a sodium deoxycholate lysis buffer and a single detergent cleanup step after peptide labeling, followed by quick off-line fractionation and MS2 analysis. We showcase the applicability of the workflow in a panel of colorectal cancer cell lines and by performing target discovery for a set of molecular glue degraders in different cell lines, in a 96-sample assay. Using this workflow, we report frequently dysregulated proteins in colorectal cancer cells and uncover cell-dependent protein degradation profiles of seven cereblon E3 ligase modulators (CRL4(CRBN)). Overall, SimPLIT is a robust method that can be easily implemented in any proteomics laboratory for medium-to-large scale TMT-based studies for deep profiling of cell lines.
      原文链接:
    • NSTL
    • Amer Chemical Soc

    Distinct Histone Post-translational Modifications during Plasmodium falciparum Gametocyte Development

    Cui, LiwangMiao, JunShrestha, SonyLucky, Amuza Byaruhanga...
    11页
    查看更多>>摘要：Histones are the building units of nucleosomes, which constitute chromatin. Histone post-translational modifications (PTMs) play an essential role in epigenetic gene regulation. The Plasmodium falciparum genome encodes canonical and variant histones and a collection of conserved enzymes for histone PTMs and chromatin remodeling. Herein, we profiled the P. falciparum histone PTMs during the development of gametocytes, the obligatory stage for parasite transmission. Mass spectrometric analysis of histones extracted from the early, middle, and late stages of gametocytes identified 457 unique histone peptides with 90 PTMs, of which 50% were novel. The gametocyte histone PTMs display distinct patterns from asexual stages, with many new methylation sites in histones H3 and H3.3 (e.g., K14, K18, and K37). Quantitative analyses revealed a high abundance of acetylation in H3 and H4, mono-methylation of H3/H3.3 K37, and ubiquitination of H3BK112, suggesting that these PTMs play critical roles in gametocytes. Gametocyte histones also showed extensive and unique combinations of PTMs. These data indicate that the parasite harbors distinct transcription regulation mechanisms during gametocyte development and lay the foundation for further characterization of epigenetic regulation in the life cycle of the malaria parasite.
      原文链接:
    • NSTL
    • Amer Chemical Soc

    MALDI(+) FT-ICR Mass Spectrometry (MS) Combined with Machine Learning toward Saliva-Based Diagnostic Screening for COVID-19

    Motta, Larissa C.Folli, Gabriely S.Marcarini, Wena D.Costa, Camila A. ....
    8页
    查看更多>>摘要：Rapid identification of existing respiratory viruses in biological samples is of utmost importance in strategies to combat pandemics. Inputting MALDI FT-ICR MS (matrix-assisted laser desorption/ionization Fourier-transform ion cyclotron resonance mass spectrometry) data output into machine learning algorithms could hold promise in classifying posi t i v e samples for SARS-CoV-2. This study aimed to develop a fast and effective methodology to perform saliva-based screening of patients with suspected COVID-19, using the MALDI FT-ICR MS technique with a support vector machine (SVM). In the method optimization, the best sample preparation was obtained with the digestion of saliva in 10 mu L of trypsin for 2 h and the MALDI analysis, which presented a satisfactory resolution for the analysis with 1 M. S V M models were created w i t h data from the analysis of 97 samples that were designated as SARS-CoV-2 positives versus 52 negatives, confirmed by RT-PCR tests. SVM1 and SVM2 models showed the best results. The calibration group obtained 100% accuracy, and the test group 95.6% (SVM1) and 86.7% (SVM2). SVM1 selected 780 variables and has a false negative rate (FNR) of 0%, while SVM2 selected only two variables with a FNR of 3%. The proposed methodology suggests a promising tool to aid screening for COVID-19.
      原文链接:
    • NSTL
    • Amer Chemical Soc

    Glycopattern Alteration of Glycoproteins in Gastrointestinal Cancer Cell Lines and Their Cell-Derived Exosomes

    Qin, YannanJiang, QiuyuLi, FangJing, Xintao...
    18页
    查看更多>>摘要：ABSTRACT: Gastrointestinal (GI) cancers constitute the largest portion of all human cancers, and the most prevalent GI cancers in China are colorectal cancer (CRC), gastric cancer (GC), and hepatocellular carcinoma (HCC). Exosomes are nanosized vesicles containing proteins, lipids, glycans, and nucleic acid, which play important roles in the tumor microenvironment and progression. Aberrant glycosylation is closely associated with GI cancers; however, little is known about the glycopattern of the exosomes from GI cancer cells. In this study, glycopatterns of HCC, CRC, and GC cell lines and their exosomes were detected using lectin microarrays. For all exosomes, (GlcNAc beta 1-4)n and Gal beta 1-4GlcNAc (DSA) were the most abundant glycans, but alpha GalNAc and alpha Gal (GSL-II and SBA) were the least. Different cancers had various characteristic glycans in either cells or exosomes. Glycans altered in cell-derived exosomes were not always consistent with the host cells in the same cancer. However, Fuc alpha 1-6GlcNAc (core fucose) and Fuc alpha 1-3(Gal beta 1-4)GlcNAc (AAL) were altered consistently in cells and exosomes although they were decreased in HCC and CRC but increased in GC. The study drew the full-scale glycan fingerprint of cells and exosomes related to GI cancer, which may provide useful information for finding specific biomarkers and exploring the underlying mechanism of glycosylation in exosomes.
      原文链接:
    • NSTL
    • Amer Chemical Soc

    Panoramic Perspective on Human Phosphosites

    Vandermarliere, ElienVranken, Wim F.Martens, LennartRamasamy, Pathmanaban...
    22页
    查看更多>>摘要：Protein phosphorylation is the most common reversible post-translational modification of proteins and is key in the regulation of many cellular processes. Due to this importance, phosphorylation is extensively studied, resulting in the availability of a large amount of mass spectrometry-based phospho-proteomics data. Here, we leverage the information in these large-scale phospho-proteomics data sets, as contained in Scop3P, to analyze and characterize proteome-wide protein phosphorylation sites (P sites). First, we set out to differentiate correctly observed P-sites from false-positive sites using five complementary site properties. We then describe the context of these P-sites in terms of the protein structure, solvent accessibility, structural transitions and disorder, and biophysical properties. We also investigate the relative prevalence of disease-linked mutations on and around P-sites. Moreover, we assess the structural dynamics of P-sites in their phosphorylated and unphosphorylated states. As a result, we show how large-scale reprocessing of available proteomics experiments can enable a more reliable view on proteome-wide P-sites. Furthermore, adding the structural context of proteins around P-sites helps uncover possible conformational switches upon phosphorylation. Moreover, by placing sites in different biophysical contexts, we show the differential preference in protein dynamics at phosphorylated sites when compared to the nonphosphorylated counterparts.
      原文链接:
    • NSTL
    • Amer Chemical Soc

    A Cytological Atlas of the Human Liver Proteome from PROTEOMESKY-LIVERHu 2.0, a Publicly Available Database

    Chen, XinguoJiang, YingRen, LiangliangLao, Yuanxiang...
    14页
    查看更多>>摘要：The liver plays a unique role as a metabolic center of the body, and also performs other important functions such as detoxification and immune response. Here, we establish a cell type-resolved healthy human liver proteome including hepatocytes (HCs), hepatic stellate cells (HSCs), Kupffer cells (KCs), and liver sinusoidal endothelial cells (LSECs) by high-resolution mass spectrometry. Overall, we quantify total 8354 proteins for four cell types and over 6000 proteins for each cell type. Analysis of this data set and regulatory pathway reveals the cellular labor division in the human liver follows the pattern that parenchymal cells make the main components of pathways, but nonparenchymal cells trigger these pathways. Human liver cells show some novel molecular features: HCs maintain KCs and LSECs homeostasis by producing cholesterol and ketone bodies; HSCs participate in xenobiotics metabolism as an agent deliverer; KCs and LSECs mediate immune response through MHC class II-TLRs and MHC class I-TGF beta cascade, respectively; and KCs play a central role in diurnal rhythms regulation through sensing diurnal IGF and temperature flux. Together, this work expands our understandings of liver physiology and provides a useful resource for future analyses of normal and diseased livers.
      原文链接:
    • NSTL
    • Amer Chemical Soc

    Novel Combined Enzymatic Approach to Analyze Nonsialylated N-Linked Glycans through MALDI Imaging Mass Spectrometry

    DelaCourt, Andrew T.Liang, HongyanDrake, Richard R.Angel, Peggi M....
    9页
    查看更多>>摘要：Alterations to N-glycan expression are relevant to the progression of various diseases, particularly cancer. In many cases, specific N-glycan structural features such as sialylation, fucosylation, and branching are of specific interest. A novel MALDI imaging mass spectrometry workflow has been recently developed to analyze these features of N-glycosylation through the utilization of endoglycosidase enzymes to cleave N-glycans from associated glycoproteins. Enzymes that have previously been utilized to cleave N-glycans include peptide-N-glycosidase F (PNGase F) to target N-glycans indiscriminately and endoglycosidase F3 (Endo F3) to target core fucosylated N-glycans. In addition to these endoglycosidases, additional N-glycan cleaving enzymes could be used to target specific structural features. Sialidases, also termed neuraminidases, are a family of enzymes that remove terminal sialic acids from glycoconjugates. This work aims to utilize sialidase, in conjunction with PNGase F/Endo F3, to enzymatically remove sialic acids from N-glycans in an effort to increase sensitivity for nonsialylated N-glycan MALDI-IMS peaks. Improving detection of nonsialylated N-glycans allows for a more thorough analysis of specific structural features such as fucosylation or branching, particularly of low abundant structures. Sialidase utilization in MALDI-IMS dramatically increases sensitivity and increases on-tissue endoglycosidase efficiency, making it a very useful companion technique to specifically detect nonsialylated N-glycans.
      原文链接:
    • NSTL
    • Amer Chemical Soc

    Identification of Microproteins in Saccharomyces cerevisiae under Different Stress Conditions

    Sun, YanHuang, JiangmeiWang, ZhiweiPan, Ni...
    9页
    查看更多>>摘要：Small open reading frame-encoded peptides (SEPs) are microproteins with a length of 100 amino acids or less, which may play a critical role in maintaining cell homeostasis under stress. Therefore, we used mass spectrometry-based proteomics to explore microproteins potentially involved in cellular stress responses in Saccharomyces cerevisiae. A total of 225 microproteins with 1920 unique peptides were identified under six culture conditions: normal, oxidation, starvation, ultraviolet radiation, heat shock, and heat shock with starvation. Among these microproteins, we found 70 SEPs with 75 unique peptides. The annotated microproteins are involved in stress-related processes, such as cell redox reactions, cell wall modification, protein folding and degradation, and DNA damage repair. It suggests that SEPs may also play similar functions under stress conditions. For example, SEP IP_008057, translated from a short coding sequence of YJL159W, may play a role in heat shock. This study identified stress-responsive SEPs in S. cerevisiae and provided valuable information to determine the functions of these proteins, which enrich the genome and proteome of S. cerevisiae and show clues to improving the stress tolerance of S. cerevisiae.
      原文链接:
    • NSTL
    • Amer Chemical Soc