Comparison of MS2, synchronous precursor selection MS3, and real-time search MS3 methodologies for lung proteomes of hydrogen sulfide treated swine

Fu, Qin; Liu, Zhen; Bhawal, Ruchika; Anderson, Elizabeth T.; Sherwood, Robert; Yang, Yong; Thannhauser, Theodore W.; Schroyen, Martine; Tang, Xiangfang; Zhang, Hongfu; Zhang, Sheng

doi:10.1007/s00216-020-03009-5

Cited by 8 publications

(10 citation statements)

References 36 publications

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“…However, 1242 proteins could be identified in MS2 mode while only 886 proteins (71%) were detected with MS3. Both observations are in line with previous studies [ 33 , 34 ].…”

Section: Resultssupporting

confidence: 94%

Optimized sample preparation and data analysis for TMT proteomic analysis of cerebrospinal fluid applied to the identification of Alzheimer’s disease biomarkers

et al. 2022

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Background Cerebrospinal fluid (CSF) is an important biofluid for biomarkers of neurodegenerative diseases such as Alzheimer’s disease (AD). By employing tandem mass tag (TMT) proteomics, thousands of proteins can be quantified simultaneously in large cohorts, making it a powerful tool for biomarker discovery. However, TMT proteomics in CSF is associated with analytical challenges regarding sample preparation and data processing. In this study we address those challenges ranging from data normalization over sample preparation to sample analysis. Method Using liquid chromatography coupled to mass-spectrometry (LC–MS), we analyzed TMT multiplex samples consisting of either identical or individual CSF samples, evaluated quantification accuracy and tested the performance of different data normalization approaches. We examined MS2 and MS3 acquisition strategies regarding accuracy of quantification and performed a comparative evaluation of filter-assisted sample preparation (FASP) and an in-solution protocol. Finally, four normalization approaches (median, quantile, Total Peptide Amount, TAMPOR) were applied to the previously published European Medical Information Framework Alzheimer’s Disease Multimodal Biomarker Discovery (EMIF-AD MBD) dataset. Results The correlation of measured TMT reporter ratios with spiked-in standard peptide amounts was significantly lower for TMT multiplexes composed of individual CSF samples compared with those composed of aliquots of a single CSF pool, demonstrating that the heterogeneous CSF sample composition influences TMT quantitation. Comparison of TMT reporter normalization methods showed that the correlation could be improved by applying median- and quantile-based normalization. The slope was improved by acquiring data in MS3 mode, albeit at the expense of a 29% decrease in the number of identified proteins. FASP and in-solution sample preparation of CSF samples showed a 73% overlap in identified proteins. Finally, using optimized data normalization, we present a list of 64 biomarker candidates (clinical AD vs. controls, p < 0.01) identified in the EMIF-AD cohort. Conclusion We have evaluated several analytical aspects of TMT proteomics in CSF. The results of our study provide practical guidelines to improve the accuracy of quantification and aid in the design of sample preparation and analytical protocol. The AD biomarker list extracted from the EMIF-AD cohort can provide a valuable basis for future biomarker studies and help elucidate pathogenic mechanisms in AD.

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“…However, 1242 proteins could be identified in MS2 mode while only 886 proteins (71%) were detected with MS3. Both observations are in line with previous studies [ 33 , 34 ].…”

Section: Resultssupporting

confidence: 94%

Optimized sample preparation and data analysis for TMT proteomic analysis of cerebrospinal fluid applied to the identification of Alzheimer’s disease biomarkers

et al. 2022

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“…Current technological advancements in proteomics have made biomarker discovery for ALS and PD more efficient. Short gradients with novel LC systems [ 120 ] provide accurate and rapid quantitation without a loss of identifications with the new RTS-MS3 quantitation [ 121 ] or BoxCar DIA [ 31 ] to gain depth, range, and completeness, while addressing some of the dynamic range challenges in biofluids makes mass spectrometry a powerful and indispensable tool in biomarker discovery.…”

Section: Discussionmentioning

confidence: 99%

Biomarkers in Neurodegenerative Diseases: Proteomics Spotlight on ALS and Parkinson’s Disease

Raghunathan

Turajane

Wong

2022

IJMS

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Neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Parkinson’s disease (PD) are both characterized by pathogenic protein aggregates that correlate with the progressive degeneration of neurons and the loss of behavioral functions. Both diseases lack biomarkers for diagnosis and treatment efficacy. Proteomics is an unbiased quantitative tool capable of the high throughput quantitation of thousands of proteins from minimal sample volumes. We review recent proteomic studies in human tissues, plasma, cerebrospinal fluid (CSF), and exosomes in ALS and PD that identify proteins with potential utility as biomarkers. Further, we review disease-related post-translational modifications in key proteins TDP43 in ALS and α-synuclein in PD studies, which may serve as biomarkers. We compare relative and absolute quantitative proteomic approaches in key biomarker studies in ALS and PD and discuss recent technological advancements which may identify suitable biomarkers for the early-diagnosis treatment efficacy of these diseases.

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“…Proteins were digested and labeled according to Thermo Fisher Scientific’s TMTpro Mass Tagging Kits and Reagents protocol (Lot number: VL313890) with slight modifications ( 48 , 49 , 50 , 51 ). A total of 25 μg protein of each sample were suspended in 50 mM triethylammonium bicarbonate (TEAB) pH 8.5, 6M urea, 2M thiourea, 1% SDS and reduced with 10 mM Tris(2-carboxyethyl) phosphine for 1 h at 34 °C, alkylated with 20 mM iodoacetamide for 45 min in the dark, and then quenched with a final concentration of 32 mM DTT.…”

Section: Methodsmentioning

confidence: 99%

“…Labeling incorporation was checked using Orbitrap Eclipse Thermo Fisher Scientific). The eluted tryptic peptides were evaporated to dryness and fractionated using a Dionex UltiMate 3000 HPLC system with the built-in micro fraction collection option in its autosampler and UV detection (Thermo Fisher Scientific) as described ( 48 , 49 , 50 , 51 ). The TMT-labeled tryptic peptides were reconstituted in buffer A (20 mM ammonium formate pH 9.5 in water) and loaded onto an XTerra MS C18 column (3.5 μm, 2.1x 150 mm) from Waters, with 20 mM ammonium formate (NH 4 FA), pH 9.5 as buffer A and 80% ACN/20% 20 mM NH 4 FA as buffer B.…”

Section: Methodsmentioning

confidence: 99%

“…External calibration for FT, IT, and quadrupole mass analyzers was performed. Raw MS data files for all the fractions were acquired using a real-time search (RTS) synchronous precursor selection MS 3 workflow as reported previously ( 48 ). Specifically, the RTS MS 3 workflow consisted of 2.5 s “Top Speed” data-dependent collisionally induced dissociation-MS/MS scans (for peptide identifications by RTS) that enabled to trigger synchronous precursor selection of 10 MS 2 product ions for subsequent MS 3 in FT.…”

Section: Methodsmentioning

confidence: 99%

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MceG stabilizes the Mce1 and Mce4 transporters in Mycobacterium tuberculosis

Fieweger

Wilburn

Montague

et al. 2023

Journal of Biological Chemistry

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Comparison of MS2, synchronous precursor selection MS3, and real-time search MS3 methodologies for lung proteomes of hydrogen sulfide treated swine

Cited by 8 publications

References 36 publications

Optimized sample preparation and data analysis for TMT proteomic analysis of cerebrospinal fluid applied to the identification of Alzheimer’s disease biomarkers

Optimized sample preparation and data analysis for TMT proteomic analysis of cerebrospinal fluid applied to the identification of Alzheimer’s disease biomarkers

Biomarkers in Neurodegenerative Diseases: Proteomics Spotlight on ALS and Parkinson’s Disease

MceG stabilizes the Mce1 and Mce4 transporters in Mycobacterium tuberculosis

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