Robust, reproducible and quantitative analysis of thousands of proteomes by micro-flow LC–MS/MS

Bian, Yangyang; Zheng, Rujie; Bayer, Florian; Wong, Cassandra; Chang, Yun-Chien; Meng, Chen; Zolg, Daniel Paul; Reinecke, Maria; Zecha, Jana; Wiechmann, Svenja; Heinzlmeir, S.; Scherr, Johannes; Hemmer, Bernhard; Baynham, Mike; Gingras, Anne‐Claude; Boychenko, Alexander; Küster, Bernhard

doi:10.1038/s41467-019-13973-x

Cited by 244 publications

(253 citation statements)

References 54 publications

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“…With an everincreasing understanding of the power (and resolution) of the method, the number of potential applications is likely to grow. In addition, with technological advancements, such as the introduction of novel isobaric labelling chemistry 12 and LC-MS throughput 13 it becomes possible to analyze large number of compounds and unravel common or distinct pathways activation.…”

Section: Resultsmentioning

confidence: 99%

In-depth characterization of Staurosporine induced proteome thermal stability changes

Chernobrovkin

Lengqvist

Körner

et al. 2020

Preprint

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Cellular thermal shift assay (CETSA) coupled with high-resolution mass-spectrometry (MS) has proven to be indispensable tool to track thermal stability changes in cellular proteins caused by various external or internal perturbations. One of the major applications of CETSA MS is still binding profile characterization of small molecule drugs. Before applying the method for characterization of novel compound it is crucially important to understand the limitations, sensitivity and ways to improve the throughput of the different method implementations. Here we present deep comprehensive profiling of Staurosporine induced proteome thermal stability alteration utilizing different experiment layouts. By applying unbiased straightforward sample preparation and data analysis approaches we were able to compare and benchmark different experiment layouts for detecting compound-induced protein stability changes in CETSA MS experiments.

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Section: Resultsmentioning

confidence: 99%

In-depth characterization of Staurosporine induced proteome thermal stability changes

Chernobrovkin

Lengqvist

Körner

et al. 2020

Preprint

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“…those that have low sample amounts available, scanning SWATH can equally be coupled to conventional proteomic nanoflow or microflow chromatography, and profit from their high sensitivity on low sample amounts. Indeed, also microflow chromatography has recently been adapted to achieve decent throughput in proteomic experiments 16,17 . Finally, we would like to discuss that scanning SWATH requires a fast qTOF mass spectrometer, but does otherwise not depend on expensive or proprietary reagents, and all of our software is freely available and easy to use (i.e.…”

Section: Discussionmentioning

confidence: 99%

“…Further, fast, efficient and robust chromatographic separations have been achieved by replacing nanoflow LC, as traditionally used in proteomics 13,14 , with setups that use higher flow-rates. This ranges from microflow LC systems (5-50 µ l/min) [15][16][17] , to novel LC devices with preformed gradients 18,19 . More recently, we have introduced proteome experiments that make use of high-flow liquid chromatography (800 µl/min).…”

Section: Introductionmentioning

confidence: 99%

Scanning SWATH acquisition enables high-throughput proteomics with chromatographic gradients as fast as 30 seconds

Messner

Demichev

Bloomfield

et al. 2019

Preprint

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Rapidly emerging applications in data-driven biology and personalised medicine call for the development of fast and reliable proteomic methods. However, the use of fast chromatographic gradients is limited by the mass spectrometers' sampling rate and signal interferences. Here we present scanningSWATH, a data-independent acquisition method, in which the DIA-typical stepwise windowed acquisition is replaced by continuous scanning with the first quadrupole.ScanningSWATH enables ultra-fast duty cycles as well as to assign precursor masses to the MS2 fragment traces. Furthermore, we have implemented the support for scanningSWATH in DIA-NN, a fully-automated software suite designed to deconvolute fast DIA experiments, which corrects for signal interferences. We show that the combination of scanningSWATH and DIA-NN enables the efficient application of high-flow liquid chromatography (800 µL/min flow rate) to proteomics. High-flow scanningSWATH increases proteomic sample throughput to the minute-scale, while the use of high-flow chromatography hardware improves reliability and robustness. Benchmarking on yeast and human cell lysates, we demonstrate that the proteomic depth achieved with five-minute high-flow scanningSWATH gradients is comparable to that obtained with several times slower nano-and microflow chromatographic gradients, even if compared to most recent studies that used microflow-SWATH or Evosep-DIA methods. The combination of scanningSWATH, advanced data processing, and industry-standard high-flow LC hardware paves a way for a new generation of cheaper and highly consistent proteomic methods. These allow the recording of hundreds of precise quantitative proteomes per day on a single instrument.

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“…In addition, due to the high reactivity of the label, these excess materials are often considered unsuitable for use following rehydration and are typically discarded as waste. 11 Alternative approaches to NanoLC are gaining in popularity, with multiple studies using capillary zone electrophoresis 14,15 and microflow chromatography 16,17 demonstrating promise as alternative methodologies. Recent work has also described the use of "standard flow" proteomics, reaching the conclusion that flow rates in the 50 -200 µl per minute range can provide quality proteomics data with proper optimization.…”

Section: Abstract Graphicmentioning

confidence: 99%

Standard Flow Multiplexed Proteomics (SFloMPro). An Accessible and Cost-Effective Alternative to NanoLC Workflows

Jenkins

Orsburn

2020

Preprint

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Multiplexed proteomics using isobaric tagging allows for simultaneously comparing the proteomes of multiple samples. In this technique, digested peptides from each sample are labeled with a chemical tag prior to pooling sample for LC-MS/MS with nanoflow chromatography (NanoLC). The isobaric nature of the tag prevents deconvolution of samples until fragmentation liberates the isotopically labeled reporter ions. To ensure efficient peptide labeling, large concentrations of labeling reagents are included in the reagent kits to allow scientists to use high ratios of chemical label per peptide. The increasing speed and sensitivity of mass spectrometers has reduced the peptide concentration required for analysis, leading to most of the label or labeled sample to be discarded. In conjunction, improvements in the speed of sample loading, reliable pump pressure, and stable gradient construction of analytical flow HPLCs has continued to improve the sample delivery process to the mass spectrometer. In this study we describe a method for performing multiplexed proteomics without the use of NanoLC by using offline fractionation of labeled peptides followed by rapid "standard flow" HPLC gradient LC-MS/MS. Standard Flow Multiplexed Proteomics (SFloMPro) enables high coverage quantitative proteomics of up to 16 mammalian samples in about 24 hours. In this study, we compare NanoLC and SFloMPro analysis of fractionated samples. Our results demonstrate that comparable data is obtained by injecting 20 µg of labeled peptides per fraction with SFloMPro, compared to 1 µg per fraction with NanoLC. We conclude that, for experiments where protein concentration is not strictly limited, SFloMPro is a competitive approach to traditional NanoLC workflows with improved up-time, reliability and at a lower relative cost per sample.Data are available via ProteomeXchange with identifier PXD016704.

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Robust, reproducible and quantitative analysis of thousands of proteomes by micro-flow LC–MS/MS

Cited by 244 publications

References 54 publications

In-depth characterization of Staurosporine induced proteome thermal stability changes

In-depth characterization of Staurosporine induced proteome thermal stability changes

Scanning SWATH acquisition enables high-throughput proteomics with chromatographic gradients as fast as 30 seconds

Standard Flow Multiplexed Proteomics (SFloMPro). An Accessible and Cost-Effective Alternative to NanoLC Workflows

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