scholarly journals A Compact Quadrupole-Orbitrap Mass Spectrometer with FAIMS Interface Improves Proteome Coverage in Short LC Gradients

2019 ◽  
Author(s):  
Dorte B. Bekker-Jensen ◽  
Ana Martínez del Val ◽  
Sophia Steigerwald ◽  
Patrick Rüther ◽  
Kyle Fort ◽  
...  
Keyword(s):  
Mass Spectrometer ◽  
Ion Mobility ◽  
Rat Tissues ◽  
Label Free ◽  
Mass Spectrometers ◽  
Proteome Coverage ◽  
Data Independent Acquisition ◽  
Orbitrap Mass Spectrometer ◽  
Peptide Precursors ◽  
Free Quantification

ABSTRACTState-of-the-art proteomics-grade mass spectrometers can measure peptide precursors and their fragments with ppm mass accuracy at sequencing speeds of tens of peptides per second with attomolar sensitivity. Here we describe a compact and robust quadrupole-orbitrap mass spectrometer equipped with a front-end High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) Interface. The performance of the Orbitrap Exploris 480 mass spectrometer is evaluated in data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes in combination with FAIMS. We demonstrate that different compensation voltages (CVs) for FAIMS are optimal for DDA and DIA, respectively. Combining DIA with FAIMS using single CVs, the instrument surpasses 2500 unique peptides identified per minute. This enables quantification of >5000 proteins with short online LC gradients delivered by the Evosep One LC system allowing acquisition of 60 samples per day. The raw sensitivity of the instrument is evaluated by analyzing 5 ng of a HeLa digest from which >1000 proteins were reproducibly identified with 5 minute LC gradients using DIA-FAIMS. To demonstrate the versatility of the instrument we recorded an organ-wide map of proteome expression across 12 rat tissues quantified by tandem mass tags and label-free quantification using DIA with FAIMS to a depth of >10,000 proteins.

scholarly journals A Compact Quadrupole-Orbitrap Mass Spectrometer with FAIMS Interface Improves Proteome Coverage in Short LC Gradients

2020 ◽  
Vol 19 (4) ◽  
pp. 716-729 ◽  
Author(s):  
Dorte B. Bekker-Jensen ◽  
Ana Martínez-Val ◽  
Sophia Steigerwald ◽  
Patrick Rüther ◽  
Kyle L. Fort ◽  
...  
Keyword(s):  
Mass Spectrometer ◽  
Ion Mobility ◽  
Rat Tissues ◽  
Label Free ◽  
Mass Spectrometers ◽  
Proteome Coverage ◽  
Data Independent Acquisition ◽  
Orbitrap Mass Spectrometer ◽  
Peptide Precursors ◽  
Free Quantification

State-of-the-art proteomics-grade mass spectrometers can measure peptide precursors and their fragments with ppm mass accuracy at sequencing speeds of tens of peptides per second with attomolar sensitivity. Here we describe a compact and robust quadrupole-orbitrap mass spectrometer equipped with a front-end High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) Interface. The performance of the Orbitrap Exploris 480 mass spectrometer is evaluated in data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes in combination with FAIMS. We demonstrate that different compensation voltages (CVs) for FAIMS are optimal for DDA and DIA, respectively. Combining DIA with FAIMS using single CVs, the instrument surpasses 2500 peptides identified per minute. This enables quantification of >5000 proteins with short online LC gradients delivered by the Evosep One LC system allowing acquisition of 60 samples per day. The raw sensitivity of the instrument is evaluated by analyzing 5 ng of a HeLa digest from which >1000 proteins were reproducibly identified with 5 min LC gradients using DIA-FAIMS. To demonstrate the versatility of the instrument, we recorded an organ-wide map of proteome expression across 12 rat tissues quantified by tandem mass tags and label-free quantification using DIA with FAIMS to a depth of >10,000 proteins.

Label-free quantification in ion mobility–enhanced data-independent acquisition proteomics

2016 ◽  
Vol 11 (4) ◽  
pp. 795-812 ◽  
Author(s):  
Ute Distler ◽  
Jörg Kuharev ◽  
Pedro Navarro ◽  
Stefan Tenzer
Keyword(s):  
Ion Mobility ◽  
Label Free ◽  
Label Free Quantification ◽  
Data Independent Acquisition ◽  
Free Quantification

Label-Free Quantification by Data Independent Acquisition Mass Spectrometry to Map Cardiovascular Proteomes

2016 ◽  
pp. 227-245
Author(s):  
Sarah J. Parker ◽  
Ronald J. Holewinski ◽  
Irina Tchernyshyov ◽  
Vidya Venkatraman ◽  
Laurie Parker ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Label Free ◽  
Label Free Quantification ◽  
Data Independent Acquisition ◽  
Free Quantification

scholarly journals Ranking Fragment Ions Based on Outlier Detection for Improved Label-Free Quantification in Data-Independent Acquisition LC–MS/MS

2015 ◽  
Vol 14 (11) ◽  
pp. 4581-4593 ◽  
Author(s):  
Aivett Bilbao ◽  
Ying Zhang ◽  
Emmanuel Varesio ◽  
Jeremy Luban ◽  
Caterina Strambio-De-Castillia ◽  
...  
Keyword(s):  
Outlier Detection ◽  
Label Free ◽  
Label Free Quantification ◽  
Data Independent Acquisition ◽  
Fragment Ions ◽  
Free Quantification

scholarly journals Fast Quantitative Analysis of timsTOF PASEF Data with MSFragger and IonQuant

2020 ◽  
Vol 19 (9) ◽  
pp. 1575-1585 ◽  
Author(s):  
Fengchao Yu ◽  
Sarah E. Haynes ◽  
Guo Ci Teo ◽  
Dmitry M. Avtonomov ◽  
Daniel A. Polasky ◽  
...  
Keyword(s):  
Ion Mobility ◽  
Peptide Identification ◽  
Label Free ◽  
Post Translational Modification ◽  
Data Set ◽  
Mobility Separation ◽  
Major Bottleneck ◽  
Ion Mobility Separation ◽  
Gas Phase Fragmentation ◽  
Free Quantification

Ion mobility brings an additional dimension of separation to LC–MS, improving identification of peptides and proteins in complex mixtures. A recently introduced timsTOF mass spectrometer (Bruker) couples trapped ion mobility separation to TOF mass analysis. With the parallel accumulation serial fragmentation (PASEF) method, the timsTOF platform achieves promising results, yet analysis of the data generated on this platform represents a major bottleneck. Currently, MaxQuant and PEAKS are most used to analyze these data. However, because of the high complexity of timsTOF PASEF data, both require substantial time to perform even standard tryptic searches. Advanced searches (e.g. with many variable modifications, semi- or non-enzymatic searches, or open searches for post-translational modification discovery) are practically impossible. We have extended our fast peptide identification tool MSFragger to support timsTOF PASEF data, and developed a label-free quantification tool, IonQuant, for fast and accurate 4-D feature extraction and quantification. Using a HeLa data set published by Meier et al. (2018), we demonstrate that MSFragger identifies significantly (∼30%) more unique peptides than MaxQuant (1.6.10.43), and performs comparably or better than PEAKS X+ (∼10% more peptides). IonQuant outperforms both in terms of number of quantified proteins while maintaining good quantification precision and accuracy. Runtime tests show that MSFragger and IonQuant can fully process a typical two-hour PASEF run in under 70 min on a typical desktop (6 CPU cores, 32 GB RAM), significantly faster than other tools. Finally, through semi-enzymatic searching, we significantly increase the number of identified peptides. Within these semi-tryptic identifications, we report evidence of gas-phase fragmentation before MS/MS analysis.

In-depth evaluation of software tools for data-independent acquisition based label-free quantification

PROTEOMICS ◽  
2015 ◽  
Vol 15 (18) ◽  
pp. 3140-3151 ◽  
Author(s):  
Jörg Kuharev ◽  
Pedro Navarro ◽  
Ute Distler ◽  
Olaf Jahn ◽  
Stefan Tenzer
Keyword(s):  
Software Tools ◽  
Label Free ◽  
Label Free Quantification ◽  
Data Independent Acquisition ◽  
Free Quantification

scholarly journals Advancing Urinary Protein Biomarker Discovery by Data-Independent Acquisition on a Quadrupole-Orbitrap Mass Spectrometer

2015 ◽  
Vol 14 (11) ◽  
pp. 4752-4762 ◽  
Author(s):  
Jan Muntel ◽  
Yue Xuan ◽  
Sebastian T. Berger ◽  
Lukas Reiter ◽  
Richard Bachur ◽  
...  
Keyword(s):  
Mass Spectrometer ◽  
Biomarker Discovery ◽  
Urinary Protein ◽  
Protein Biomarker ◽  
Data Independent Acquisition ◽  
Orbitrap Mass Spectrometer

scholarly journals Parallel accumulation – serial fragmentation combined with data-independent acquisition (diaPASEF): Bottom-up proteomics with near optimal ion usage

2019 ◽  
Author(s):  
Florian Meier ◽  
Andreas-David Brunner ◽  
Max Frank ◽  
Annie Ha ◽  
Isabell Bludau ◽  
...  
Keyword(s):  
Ion Mobility ◽  
Data Extraction ◽  
Ion Current ◽  
Proteome Coverage ◽  
Data Independent Acquisition ◽  
Quantitative Accuracy ◽  
Peptide Precursor ◽  
Mobility Device ◽  
High Degree ◽  
Very High

ABSTRACTData independent acquisition (DIA) modes isolate and concurrently fragment populations of different precursors by cycling through segments of a predefined precursor m/z range. Although these selection windows collectively cover the entire m/z range, overall only a few percent of all incoming ions are sampled. Making use of the correlation of molecular weight and ion mobility in a trapped ion mobility device (timsTOF Pro), we here devise a novel scan mode that samples up to 100% of the peptide precursor ion current. We extend an established targeted data extraction workflow by including the ion mobility dimension for both signal extraction and scoring, thereby increasing the specificity for precursor identification. Data acquired from whole proteome digests and mixed organism samples demonstrate deep proteome coverage and a very high degree of reproducibility as well as quantitative accuracy, even from 10 ng sample amounts.

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