Shotgun Protein Identification and Quantification by Mass Spectrometry

2009 ◽  
pp. 261-288 ◽  
Author(s):  
Bingwen Lu ◽  
Tao Xu ◽  
Sung Kyu Park ◽  
John R. Yates
Keyword(s):  
Mass Spectrometry ◽  
Protein Identification ◽  
Identification And Quantification

scholarly journals Ultrafast and Reproducible Proteomics from Small Amounts of Heart Tissue Enabled by Azo and timsTOF Pro

2021 ◽  
Author(s):  
Timothy J Aballo ◽  
David S Roberts ◽  
Jake A Melby ◽  
Kevin M Buck ◽  
Kyle A Brown ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Large Scale ◽  
Protein Identification ◽  
Heart Tissue ◽  
Tryptic Digestion ◽  
Bottom Up ◽  
Preparation Methods ◽  
Liquid Chromatography Tandem Mass ◽  
And Function ◽  
Identification And Quantification

Global bottom-up mass spectrometry (MS)-based proteomics is widely used for protein identification and quantification to achieve a comprehensive understanding of the composition, structure, and function of the proteome. However, traditional sample preparation methods are time-consuming, typically including overnight tryptic digestion, extensive sample clean-up to remove MS-incompatible surfactants, and offline sample fractionation to reduce proteome complexity prior to online liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Thus, there is a need for a fast, robust, and reproducible method for protein identification and quantification from complex proteomes. Herein, we developed an ultrafast bottom-up proteomics method enabled by Azo, a photocleavable, MS-compatible surfactant that effectively solubilizes proteins and promotes rapid tryptic digestion, combined with the Bruker timsTOF Pro, which enables deeper proteome coverage through trapped ion mobility spectrometry (TIMS) and parallel accumulation-serial fragmentation (PASEF) of peptides. We applied this method to analyze the complex human cardiac proteome and identified nearly 4,000 protein groups from as little as 1 mg of human heart tissue in a single one-dimensional LC-TIMS-MS/MS run with high reproducibility. Overall, we anticipate this ultrafast, robust, and reproducible bottom-up method empowered by both Azo and the timsTOF Pro will be generally applicable and greatly accelerate the throughput of large-scale quantitative proteomic studies. Raw data are available via the MassIVE repository with identifier MSV000087476.

Shotgun Protein Identification and Quantification by Mass Spectrometry in Neuroproteomics

2009 ◽  
pp. 229-259 ◽  
Author(s):  
Bingwen Lu ◽  
Tao Xu ◽  
Sung Kyu Park ◽  
Daniel B. McClatchy ◽  
Lujian Liao ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Identification ◽  
Identification And Quantification

scholarly journals Narrow Precursor Mass Range for DIA-MS Enhances Protein Identification and Quantification

2021 ◽  
Author(s):  
Huoming Zhang ◽  
Dalila Bensaddek
Keyword(s):  
Mass Spectrometry ◽  
Total Protein ◽  
Quantitative Proteomics ◽  
Protein Identification ◽  
Mass Range ◽  
Systematic Evaluation ◽  
Data Independent Acquisition ◽  
Subsequent Application ◽  
Accurate Quantification ◽  
Identification And Quantification

Data independent acquisition - mass spectrometry (DIA-MS) is becoming widely utilised for robust and accurate quantification of samples in quantitative proteomics. Here, we describe the systematic evaluation of the effects of DIA precursor mass range on total protein identification and quantification. We show that a narrow mass range of precursors (~250 m/z) for DIA-MS enables a higher number of protein identifications. Subsequent application of DIA with narrow precursor range (from 400 to 650 m/z) on Arabidopsis sample with spike-in of known proteins identified 34.7% more proteins than in conventional DIA (cDIA) with a wide precursor range of 400-1200 m/z. When combining several DIA-MS analyses with narrow precursor ranges (i.e., 400-650, 650-900 and 900-1200 m/z), we were able to quantify 10,099 protein groups with a median coefficient of variation of <6%. These findings represent a 59.4% increase in the number of proteins quantified than with cDIA analysis. This is particularly important for low abundance proteins, as exemplified by the 6-protein mix spike-in. In cDIA only 5 out of the 6-protein mix were quantified while our approach allowed accurate quantitation of all six proteins.

scholarly journals Narrow Precursor Mass Range for DIA–MS Enhances Protein Identification and Quantification in Arabidopsis

Life ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 982
Author(s):  
Huoming Zhang ◽  
Dalila Bensaddek
Keyword(s):  
Mass Spectrometry ◽  
Total Protein ◽  
Quantitative Proteomics ◽  
Protein Identification ◽  
Mass Range ◽  
Systematic Evaluation ◽  
Data Independent Acquisition ◽  
Subsequent Application ◽  
Accurate Quantification ◽  
Identification And Quantification

Data independent acquisition–mass spectrometry (DIA–MS) is becoming widely utilised for robust and accurate quantification of samples in quantitative proteomics. Here, we describe the systematic evaluation of the effects of DIA precursor mass range on total protein identification and quantification. We show that a narrow mass range of precursors (~250 m/z) for DIA–MS enables a higher number of protein identifications. Subsequent application of DIA with narrow precursor range (from 400 to 650 m/z) on an Arabidopsis sample with spike-in known proteins identified 34.7% more proteins than in conventional DIA (cDIA) with a wide precursor range of 400–1200 m/z. When combining several DIA–MS analyses with narrow precursor ranges (i.e., 400–650, 650–900 and 900–1200 m/z), we were able to quantify 10,099 protein groups with a median coefficient of variation of <6%. These findings represent a 54.7% increase in the number of proteins quantified than with cDIA analysis. This is particularly important for low abundance proteins, as exemplified by the six-protein mix spike-in. In cDIA only five out of the six-protein mix were quantified while our approach allowed accurate quantitation of all six proteins.

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