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Micro-flow LC-MS/MS Based Single Shot Proteomics

ABSTRACT: In this study, we systematically optimized the micro-flow LC-MS/MS system based ssDDA proteomics. Our results show that combination of 2 – 3 h gradient with prosit rescore method identified the highest number of proteins and peptides. There is no benefit to increase column length. A single 3 h run identified about 9,200 proteins from 50 µg of Arabidopsis protein digest. The micro-flow LC-MS/MS system based ssDDA proteomics can be widely used for routine proteomics analysis of large cohort samples.

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Homo Sapiens (human) Arabidopsis Thaliana (mouse-ear Cress) Mus Musculus (mouse)

TISSUE(S): Root, Cell Culture, Thymus, Small Intestine

SUBMITTER: Yangyang Bian

LAB HEAD: Bernhard Kuster

PROVIDER: PXD023587 | Pride | 2021-07-01

REPOSITORIES: Pride

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	1h_28Hz_loading.rar	Other
	1h_41Hz_loading.rar	Other
	200719_P3_mouse_9591_1h_100ug_R1.raw	Raw
	200719_P3_mouse_9591_1h_10ug_R1.raw	Raw
	200719_P3_mouse_9591_1h_150ug_R1.raw	Raw

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Publications

Identification of 7 000-9 000 Proteins from Cell Lines and Tissues by Single-Shot Microflow LC-MS/MS.

Bian Yangyang Y The Matthew M Giansanti Piero P Mergner Julia J Zheng Runsheng R Wilhelm Mathias M Boychenko Alexander A Kuster Bernhard B

Analytical chemistry 20210614 25

A current trend in proteomics is to acquire data in a "single-shot" by LC-MS/MS because it simplifies workflows and promises better throughput and quantitative accuracy than schemes that involve extensive sample fractionation. However, single-shot approaches can suffer from limited proteome coverage when performed by data dependent acquisition (ssDDA) on nanoflow LC systems. For applications where sample quantities are not scarce, this study shows that high proteome coverage can be obtained usin ...[more]

PMID: 34124897

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Comparision of Nano and Micro-flow LC-MS/MS Based Single Shot Proteomics

Project description:In this study, we systematically optimized the micro-flow LC-MS/MS system based ssDDA proteomics. Our results show that combination of 2 – 3 h gradient with prosit rescore method identified the highest number of proteins and peptides. There is no benefit to increase column length. A single 3 h run identified about 9,200 proteins from 50 µg of Arabidopsis protein digest. The micro-flow LC-MS/MS system based ssDDA proteomics can be widely used for routine proteomics analysis of large cohort samples. We compared our micro data with the state of art nano LC data.

2021-07-01 | PXD024169 | Pride

Robust micro-flow LC-MS/MS for proteome analysis – 38,000 runs and counting

Project description:Micro-flow liquid chromatography tandem mass spectrometry (µLC–MS/MS) is increasingly becoming an alternative to nano-LC–MS/MS for the analysis of proteomes. We have recently demonstrated the potential of a µLC–MS/MS system operating with a 1 mm i.d. × 150 mm column and at a flow rate of 50 µl/min for high-throughput applications. Based on the analysis of ~38,000 samples measured on two instruments over the past two years, we show that the approach is extremely robust. Up to 1,500 analysis were performed within one month and >14,000 samples could be analyzed on a single column without loss of chromatographic performance. Samples included proteomes of cell lines, animal tissue and human body fluids, which were analyzed either with or without prior peptide fractionation or stable isotope labeling. We show that the µLC–MS/MS system is capable of measuring ~2,700 proteins from human plasma and ~5,200 proteins from human urine within one day, demonstrating its potential for in-depth as well as high-throughput clinical application.

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Evaluation of disposable-trap column nanoLC-FAIMS-MS/MS for the proteomic analysis of FFPE tissue

Project description:Proteomic biomarker discovery using formalin-fixed paraffin-embedded (FFPE) tissue requires robust workflows to support the analysis of large cohorts of patient samples. It also requires finding a reasonable balance between achieving high proteomic depth and limiting overall analysis time. To this end, we evaluated the merits of online coupling of disposable trap column nano-flow liquid chromatography, high-field asymmetric-waveform ion-mobility spectrometry (FAIMS) and tandem mass spectrometry (nLC-FAIMS-MS/MS). The data shows that ≤ 600 ng of peptide digest should be loaded onto the chromatographic part of the system. Careful characterization of the FAIMS settings enabled the choice of optimal combinations of compensation voltages (CV) as a function of the employed LC gradient time. We found nLC-FAIMS-MS/MS to be on par with stage tip-based off-line high pH reversed phase fractionation in terms of proteomic depth and reproducibility of protein quantification (coefficient of variation ≤ 15% for 90 % of all proteins) but requiring 50% less sample and substantially reducing sample handling. Using FFPE material from lymph node, lung and prostate tissue as examples, we show that nLC-FAIMS-MS/MS can identify 5-6,000 proteins from the respective tissue within a total of 3 hours of analysis time.

2021-11-02 | PXD028136 | Pride

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Minor Antigens in Membranous Nephropathy Identified by Mass Spectrometry

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2023-04-06 | PXD035853 | Pride

Functional Variomics Library testing for drug resistance in yeast

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Transcription profiling of Arabidopsis callose synthase deficient (pmr4-1) mutant and wild type plants infected with powdery mildew

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