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第三届全国质谱分析学术报告会

作者： Heiner KOCH Scarlet BECK Jürgen COX Florian MEIER Markus LUBECK Oliver RAETHER Kefei WANG Max Planck Institute of Biochemistry

We recently described the parallel accumulation–serial fragmentation (PASEF) method (Figure 1)that has shown the promise to increase the sequencing speed and sensitivity of MS/MS scans on a prototype TIMS-QTOF instru... 详细信息

关键词： parallel accumulation-serial fragmentation PASEF QTOF Instrument

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Single-Cell Proteomic Characterization of Drug-Resistant Prostate Cancer Cells Reveals Molecular Signatures Associated with Morphological Changes

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MOLECULAR & CELLULAR PROTEOMICS 2025年第4期24卷 100949页

作者： Woo, Jongmin Loycano, Michael Amanullah, Md Qian, Jiang Amend, Sarah R. Pienta, Kenneth J. Zhang, Hui Johns Hopkins Univ Sch Med Dept Pathol Baltimore MD 21205 USA Johns Hopkins Univ Sch Med Dept Urol Baltimore MD 21205 USA Johns Hopkins Univ James Buchanan Brady Urol Inst Canc Ecol Ctr Sch Med Baltimore MD USA Johns Hopkins Univ Sch Med Dept Ophthalmol Baltimore MD USA Johns Hopkins Univ Sch Med Dept Oncol Baltimore MD 21205 USA

This study delves into the proteomic intricacies of drug-resistant cells (DRCs) within prostate cancer, which are known for their pivotal roles in therapeutic resistance, relapse, and metastasis. Utilizing single-cell proteomics (SCP) with an optimized high-throughput data-independent acquisition (DIA) approach with the throughput of 60 sample per day, we characterized the proteomic landscape of DRCs in comparison to parental PC3 cells. This DIA method allowed for robust and reproducible protein quantification at the single-cell level, enabling the identification and quantification of over 1300 proteins per cell on average. Distinct proteomic sub-clusters within the DRC population were identified, closely linked to variations in cell size. The study uncovered novel protein signatures, including the regulation of proteins critical for cell adhesion and metabolic processes, as well as the upregulation of surface proteins and transcription factors pivotal for cancer progression. Furthermore, by conducting single-cell RNA-seq (scRNA-seq) analysis, we identified six upregulated and 10 downregulated genes consistently altered in drug-treated cells across both SCP and scRNAseq platforms. These findings underscore the heterogeneity of DRCs and their unique molecular signatures, providing valuable insights into their biological behavior and

关键词： prostate cancer cell line drug resistant single-cell proteomics molecular signature AGC Automatic Gain Control ciRT Calibration Indexed Retention Time CPM Counts Per Million DDM n-dodecyl-ß-D-Maltoside DIA-MS Data Independent Acquisition Mass Spectrometry DRCs Drug-Resistant Cells FDR False Discovery Rate IAA Iodoacetamide KNN K-Nearest Neighbor LC-FAIMS-MS Liquid Chromatography with Field Asymmetric Ion Mobility Spectrometry Mass Spectrometry LD-PCR Long-Distance Polymerase Chain Reaction LMNG Lauryl Maltose Neopentyl Glycol PASEF parallel accumulation-serial fragmentation PCA Principal Component Analysis SCP Single-Cell Proteomics SPD Samples Per Day TCEP Tris(2-carboxyethyl)phosphine TEAB Triethylammonium Bicarbonate TIMS Trapped Ion Mobility Spectrometry UMAP Uniform Manifold Approximation and Projection

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Electrophoresis-Correlative Ion Mobility Deepens Single-Cell Proteomics in Capillary Electrophoresis Mass Spectrometry

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MOLECULAR & CELLULAR PROTEOMICS 2025年第2期24卷 100892页

作者： Shen, Bowen Zhou, Fei Nemes, Peter Univ Maryland Dept Chem & Biochem College Pk MD 20742 USA

Detection of trace-sensitive signals is a current challenge in single-cell mass spectrometry (MS) proteomics. Separation prior to detection improves the fidelity and depth of proteome identification and quantification. We recently recognized capillary electrophoresis (CE) electrospray ionization (ESI) for ordering peptides into mass-to-charge (m/z)-dependent series, introducing electrophoresis-correlative (Eco) data-independent acquisition. Here, we demonstrate that these correlations based on electrophoretic mobility (mu ef) in the liquid phase are transferred into the gas phase, essentially temporally sorting the peptide ions into charge- dependent ion mobility (IM, 1/K-0) trends (rho > 0.97). Rather than sampling the entire IM region broadly, we pursued these predictable correlations to schedule narrower frames. Compared to classical data-dependent (dda) PASEF, Eco-framing significantly enhanced the resolution of IM MS (IMS) on a trapped IM mass spectrometer (timsTOF PRO). This approach returned-50% more proteins from HeLa proteome digests approximating to one-to-two cells, identifying-962 proteins from-200 pg in <20 min of effective electrophoresis, without match-between-runs. As a proof of principle, we deployed Eco-IMS to detect 1157 proteins by analyzing <4% of the total proteome content in single, yolk-laden embryonic stem cells (-80-mu m) that were isolated from the animal cap of the South African clawed frog (Xenopus laevis). Quantitative profiling of nine different blastomeres revealed detectable differences among these cells, which are normally fated to form the ectoderm but retain pluripotentiality. Eco-framing in the IM dimension effectively deepens the proteome sensitivity in IMS using ddaPASEF, facilitating the proteome-driven classification of differentiating cells, as demonstrated in the chordate frog embryo in this report.

关键词： single cell capillary electrophoresis mass spectrometry proteomics Xenopus laevis AmBic ammonium bicarbonate ACN acetonitrile CE capillary electrophoresis DDA data-dependent acquisition DIA data-independent acquisition Eco electrophoresis-correlative ESI electrospray ionization FA formic acid FAIMS field asymmetric ion mobility spectrometry HCA hierarchical cluster analysis LC liquid chromatography LFQ label-free quantification IM ion mobility IMS IM mass spectrometry MBR match-between-run MS mass spectrometry MT migration time PASEF parallel accumulation-serial fragmentation timsTOF PRO trapped ion mobility mass spectrometer UMAP uniform manifold approximation and projection

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TIMAHAC: Streamlined Tandem IMAC-HILIC Workflow for Simultaneous and High-Throughput Plant Phosphoproteomics and N-glycoproteomics

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MOLECULAR & CELLULAR PROTEOMICS 2024年第5期23卷 100762页

作者： Chen, Chin-Wen Lin, Pei-Yi Lai, Ying-Mi Lin, Miao-Hsia Lin, Shu-Yu Hsu, Chuan-Chih Acad Sinica Inst Plant & Microbial Biol Taipei Taiwan Acad Sinica Agr Biotechnol Res Ctr Taipei Taiwan Natl Taiwan Univ Coll Med Dept Microbiol Taipei Taiwan Acad Sinica Common Mass Spectrometry Facil Prote & Prot Modifi Taipei Taiwan

Protein post-translational modifications (PTMs) are crucial in plant cellular processes, particularly in protein folding and signal transduction. N-glycosylation and phosphorylation are notably significant PTMs, playing essential roles in regulating plant responses to environmental stimuli. However, current sequential enrichment methods for simultaneous analysis of phosphoproteome and N-glycoproteome are labor-intensive and time-consuming, limiting their throughput. Addressing this challenge, this study introduces a novel tandem S-Trap-IMAC-HILIC (S- Trap: suspension trapping;IMAC: immobilized metal ion affinity chromatography;HILIC: hydrophilic interaction chromatography) strategy, termed TIMAHAC, for simultaneous analysis of plant phosphoproteomics and N-glycoproteomics. This approach integrates IMAC and HILIC into a tandem tip format, streamlining the enrichment process of phosphopeptides and N-glycopeptides. The key innovation lies in the use of a unified buffer system and an optimized enrichment sequence to enhance efficiency and reproducibility. The applicability of TIMAHAC was demonstrated by analyzing the Arabidopsis phosphoproteome and N-glycoproteome in response to abscisic acid (ABA) treatment. Up to 1954 N-glycopeptides and 11,255 phosphopeptides were identified from Arabidopsis, indicating its scalability for plant tissues. Notably, distinct perturbation patterns were observed in the phosphoproteome and N-glycoproteome, suggesting their unique contributions to ABA response. Our results reveal that TIMAHAC offers a comprehensive approach to studying complex regulatory mechanisms and PTM interplay in plant biology, paving the way for in-depth investigations into plant signaling networks.

关键词： IMAC HILIC phosphoproteomics N-glycoproteomics ABA signaling Arabidopsis thaliana AA acetic acid ABA abscisic acid ACN acetonitrile CE collision energy CV coefficient of variation DDA data-dependent acquisition FA formic acid HILIC hydrophilic interaction chromatography IM ion mobility IMAC immobilized metal ion affinity chromatography LC liquid chromatography MS mass spectrometry PASEF parallel accumulation-serial fragmentation PTM post-translational modification SD standard deviation SPD samples per day S-Trap suspension trapping TFA trifluoroacetic acid TIMS trapped ion mobility spectrometry XIC extracted ion chromatography

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Acquisition and Analysis of DIA-Based Proteomic Data: A Comprehensive Survey in 2023

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MOLECULAR & CELLULAR PROTEOMICS 2024年第2期23卷 100712页

作者： Lou, Ronghui Shui, Wenqing ShanghaiTech Univ iHuman Inst Shanghai Peoples R China ShanghaiTech Univ Sch Life Sci & Technol Shanghai Peoples R China

Data-independent acquisition (DIA) mass spectrometry (MS) has emerged as a powerful technology for high-throughput, accurate, and reproducible quantitative proteomics. This review provides a comprehensive overview of recent advances in both the experimental and computational methods for DIA proteomics, from data acquisition schemes to analysis strategies and software tools. DIA acquisition schemes are categorized based on the design of precursor isolation windows, highlighting wide-window, overlapping-window, narrow-window, scanning quadrupole-based, and parallel accumulation-serial fragmentation-enhanced DIA methods. For DIA data analysis, major strategies are classified into spectrum reconstruction, sequence-based search, library- based search, de novo sequencing, and sequencing- independent approaches. A wide array of software tools implementing these strategies are reviewed, with details on their overall workflows and scoring approaches at different steps. The generation and optimization of spectral libraries, which are critical resources for DIA analysis, are also discussed. Publicly available benchmark datasets covering global proteomics and phosphoproteomics are summarized to facilitate performance evaluation of various software tools and analysis workflows. Continued advances and synergistic developments of versatile components in DIA workflows are expected to further enhance the power of DIA-based proteomics.

关键词： bottom-up proteomics data-independent acquisition DIA data analysis spectral library benchmark data CID collision-induced dissociation DDA data-dependent acquisition DIA data-independent acquisition MS mass spectrometry PASEF parallel accumulation-serial fragmentation TOF time of flight

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Bottom-up Histone Post-translational Modification Analysis using Liquid Chromatography, Trapped Ion Mobility Spectrometry, and Tandem Mass Spectrometry

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JOURNAL OF PROTEOME RESEARCH 2024年第9期23卷 3867-3876页

作者： Fuller, Cassandra N. Tose, Lilian Valadares Vitorino, Francisca N. L. Bhanu, Natarajan V. Panczyk, Erin M. Park, Melvin A. Garcia, Benjamin A. Fernandez-Lima, Francisco Florida Int Univ Dept Chem & Biochem Miami FL 33199 USA Washington Univ Sch Med Dept Biochem & Mol Biophys St Louis MO 63110 USA Bruker Daltonics Inc Billerica MA 01821 USA Florida Int Univ Biomol Sci Inst Miami FL 33199 USA

The amino acid position within a histone sequence and the chemical nature of post-translational modifications (PTMs) are essential for elucidating the "Histone Code". Previous work has shown that PTMs induce specific biological responses and are good candidates as biomarkers for diagnostics. Here, we evaluate the analytical advantages of trapped ion mobility (TIMS) with parallel accumulation-serial fragmentation (PASEF) and tandem mass spectrometry (MS/MS) for bottom-up proteomics of model cancer cells. The study also considered the use of nanoliquid chromatography (LC) and traditional methods: LC-TIMS-PASEF-ToF MS/MS vs nLC-TIMS-PASEF-ToF MS/MS vs nLC-MS/MS. The addition of TIMS and PASEF-MS/MS increased the number of detected peptides due to the added separation dimension. All three methods showed high reproducibility and low RSD in the MS domain (<5 ppm). While the LC, nLC and TIMS separations showed small RSD across samples, the accurate mobility (1/K-0) measurements (<0.6% RSD) increased the confidence of peptide assignments. Trends were observed in the retention time and mobility concerning the number and type of PTMs (e.g., ac, me(1-3)) and their corresponding unmodified, propionylated peptide that aided in peptide assignment. Mobility separation permitted the annotation of coeluting structural and positional isomers and compared with nLC-MS/MS showed several advantages due to reduced chemical noise.

关键词： histones post-translational modifications trapped ion mobility spectrometry bottom-up proteomics tandem mass spectrometry nano liquid chromatography parallel accumulation-serial fragmentation

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Eleven shades of PASEF

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EXPERT REVIEW OF PROTEOMICS 2024年第9-10期21卷 367-376页

作者： Mendes, Marta L. Borrmann, Klara F. Dittmar, Gunnar Luxembourg Inst Hlth Dept Infect & Immun 1a Rue Thomas Edison L-1445 Strassen Luxembourg Univ Luxembourg Dept Life Sci & Med Belvaux Luxembourg

IntroductionThe introduction of trapped ion mobility spectrometry (TIMS) in combination with fast high-resolution time-of-flight (TOF) mass spectrometry to the proteomics field led to a jump in protein identifications and quantifications, as well as a lowering of the limit of detection for proteins from biological samples. parallel accumulation-serial fragmentation (PASEF) is a driving force for this development and has been adapted to discovery as well as targeted *** coveredOver the last decade, the PASEF concept has been optimized and led to the implementation of eleven new measurement techniques. In this review, we describe all currently described PASEF measurement techniques and their application to clinical proteomics. Literature was searched using PubMed and Google Scholar search *** OpinionThe use of a dual TIMS tunnel has revolutionized the depth and the speed of proteomics measurements. Currently, we witness how this technique is pushing clinical proteomics forward.

关键词： Proteomics ion mobility TIMS-TOF PASEF parallel accumulation-serial fragmentation targeted proteomics DIA

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The One Hour Human Proteome

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MOLECULAR & CELLULAR PROTEOMICS 2024年第5期23卷 100760页

作者： Serrano, Lia R. Peters-Clarke, Trenton M. Arrey, Tabiwang N. Damoc, Eugen Robinson, Margaret Lea Lancaster, Noah M. Shishkova, Evgenia Moss, Corinne Pashkova, Anna Sinitcyn, Pavel Brademan, Dain R. Quarmby, Scott T. Peterson, Amelia C. Zeller, Martin Hermanson, Daniel Stewart, Hamish Hock, Christian Makarov, Alexander Zabrouskov, Vlad Coon, Joshua J. Univ Wisconsin Madison Dept Chem Madison WI 53706 USA Univ Wisconsin Madison Dept Biomol Chem Madison WI 53706 USA Thermo Fisher Sci GmbH Bremen Germany Natl Ctr Quantitat Biol Complex Syst Madison WI 53715 USA Morgridge Inst Res Madison WI 53715 USA Thermo Fisher Sci San Jose CA USA

We describe deep analysis of the human proteome in less than 1 h. We achieve this expedited proteome characterization by leveraging state-of-the-art sample preparation, chromatographic separations, and data analysis tools, and by using the new Orbitrap Astral mass spectrometer equipped with a quadrupole mass filter, a high-field Orbitrap mass analyzer, and an asymmetric track loss- less (Astral) mass analyzer. The system offers high tandem mass spectrometry acquisition speed of 200 Hz and detects hundreds of peptide sequences per second within data-independent acquisition or data-dependent acquisition modes of operation. The fast-switching capabilities of the new quadrupole complement the sensitivity and fast ion scanning of the Astral analyzer to enable narrow-bin data-independent analysis methods. Over a 30-min active chromatographic method consuming a total analysis time of 56 min, the Q-Orbitrap-Astral hybrid MS collects an average of 4319 MS1 scans and 438,062 tandem mass spectrometry scans per run, producing 235,916 peptide sequences (1% false discovery rate). On average, each 30-min analysis achieved detection of 10,411 protein groups (1% false discovery rate). We conclude, with these results and alongside other recent reports, that the 1-h human proteome is within reach.

关键词： proteomics mass spectrometry technology development instrumentation proteome data independent acquisition single shot high throughput CRISPR ACN acetonitrile AGC automatic gain control Astral asymmetric track lossless DDA data-dependent acquisition DIA data-independent acquisition FA formic acid FDR false discovery rate GO gene ontology HRAM high-resolution accurate mass LC liquid chromatography LC-MS/MS liquid chromatography with tandem mass spectrometry LFQ label-free quantitation MP mobile phase MS mass spectrometry PASEF parallel accumulation-serial fragmentation Q-OT quadrupole-Orbitrap RSD relative standard deviation TFA trifluoroacetic acid TIMS trapped ion mobility spectrometry TOF time-of-flight

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Exploiting ion-mobility mass spectrometry for unraveling proteome complexity

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JOURNAL OF SEPARATION SCIENCE 2023年第18期46卷 e2300512页

作者： Perchepied, Stan Zhou, Zhuoheng Mitulovic, Goran Eeltink, Sebastiaan Vrije Univ Brussel VUB Dept Chem Engn Brussels Belgium Bruker Daltonics Inc Vienna Austria Pl Laan 2 B-1050 Brussels Belgium

Ion mobility spectrometry-mass spectrometry (IMS-MS) is experiencing rapid growth in proteomic studies, driven by its enhancements in dynamic range and throughput, increasing the quantitation precision, and the depth of proteome coverage. The core principle of ion mobility spectrometry is to separate ions in an inert gas under the influence of an electric field based on differences in drift time. This minireview provides an introduction to IMS operation modes and a description of advantages and limitations is presented. Moreover, the principles of trapped IMS-MS (TIMS-MS), including parallel accumulation-serial fragmentation are discussed. Finally, emerging applications linked to TIMS focusing on sample throughput (in clinical proteomics) and sensitivity (single-cell proteomics) are reviewed, and the possibilities of intact protein analysis are discussed.

关键词： clinical proteomics data-independent acquisition parallel accumulation-serial fragmentation single-cell analysis TIMS-TOF

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The Landscape and Perspectives of the Human Gut Metaproteomics

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MOLECULAR & CELLULAR PROTEOMICS 2024年第5期23卷 100763页

作者： Sun, Zhongzhi Ning, Zhibin Figeys, Daniel Univ Ottawa Fac Med Sch Pharmaceut Sci Ottawa ON Canada Univ Ottawa Fac Med Dept Biochem Microbiol & Immunol Ottawa ON Canada

The human gut microbiome is closely associated with human health and diseases. Metaproteomics has emerged as a valuable tool for studying the functionality of the gut microbiome by analyzing the entire proteins present in microbial communities. Recent advancements in liquid chromatography and tandem mass spectrometry (LC-MS/MS) techniques have expanded the detection range of metaproteomics. However, the overall coverage of the proteome in metaproteomics is still limited. While metagenomics studies have revealed substantial microbial diversity and functional potential of the human gut microbiome, few studies have summarized and studied the human gut microbiome landscape revealed with metaproteomics. In this article, we present the current landscape of human gut metaproteomics studies by reanalyzing the identification results from 15 published studies. We quantified the limited proteome coverage in metaproteomics and revealed a high proportion of annotation coverage of metaproteomics-identified proteins. We conducted a preliminary comparison between the metaproteomics view and the metagenomics view of the human gut microbiome, identifying key areas of consistency and divergence. Based on the current landscape of human gut metaproteomics, we discuss the feasibility of using metaproteomics to study functionally unknown proteins and propose a whole workflow peptide-centric analysis. Additionally, we suggest enhancing metaproteomics analysis by refining taxonomic classification and calculating confidence scores, as well as developing tools for analyzing the interaction between taxonomy and function.

关键词： metaproteomics human gut microbiome proteome coverage functional annotation CDS coding sequences COG Cluster of Orthologous Groups DDA data-dependent acquisition DIA data-independent acquisition GO Gene Ontology LCA lowest common ancestor LC-MS/MS liquid chromatography and tandem mass spectrometry PASEF parallel accumulation-serial fragmentation PTMs post-translation modifications UHGG Unified Human Gastrointestinal Genome

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