TMT Labeling for the Masses: A Robust and Cost-efficient, In-solution Labeling Approach

Zecha, Jana; Satpathy, Shankha; Kanashova, Tamara; Avanessian, Shayan C.; Kane, M. Harry; Clauser, Karl R.; Mertins, Philipp; Carr, Steven A.; Küster, Bernhard

doi:10.1074/mcp.tir119.001385

Cited by 287 publications

(267 citation statements)

References 28 publications

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“…Future efforts will also be aimed at reducing the amount of labeling reagent required for on-antibody TMT labeling to further reduce the cost of the UbiFast method. Zecha et al have recently presented a protocol for TMT labeling that reduces the quantity of required labeling reagent by reducing reaction volumes 31 .…”

Section: Discussionmentioning

confidence: 99%

UbiFast, a rapid and deep-scale ubiquitylation profiling approach for biology and translational research

Udeshi

Mani

Satpathy

et al. 2019

Preprint

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Protein ubiquitylation is involved in a plethora of cellular processes. Defects in the ubiquitin system are at the root of many acquired and hereditary diseases. While antibodies directed at ubiquitin remnants (K-ɛ-GG) have improved the ability to monitor ubiquitylation using mass spectrometry, methods for highly-multiplexed measurement of ubiquitylation in tissues and primary cells using sub-milligram amounts of sample remains a challenge. Here we present a highly-sensitive, rapid and multiplexed protocol for quantifying ~10,000 ubiquitylation sites from as little as 500 ug peptide per sample from cells or tissue in a TMT10 plex in ca. 5 hr. High-field Asymmetric Ion Mobility Spectrometry (FAIMS) is used to improve quantitative accuracy for posttranslational modification analysis. We use the approach to rediscover substrates of the E3 ligase targeting drug lenalidomide and to identify proteins modulated by ubiquitylation in models of basal and luminal human breast cancer. The sensitivity and speed of the UbiFast method makes it suitable for large-scale studies in primary tissue samples.

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Section: Discussionmentioning

confidence: 99%

UbiFast, a rapid and deep-scale ubiquitylation profiling approach for biology and translational research

Udeshi

Mani

Satpathy

et al. 2019

Preprint

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“…However, recent studies have demonstrated effective complete labeling of peptides with a lower ratio of peptides, with 1:4 and even 1:2 shown to be effective. 11,12 Currently, the smallest aliquot commercially available for each labeling compound is 800 µg of label per sample. This appears to constitute a large discrepancy, as the typical upper limit for peptides for NanoLC separation, is considered 0.2 -4 µg of peptide injection.…”

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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“…Samples were divided into two groups-peripheral blood-and cord blood-derived cells-such that each set could be separately labeled with TMT 6-plex isobaric mass tagging reagents (Thermo Fisher Scientific) as previously described (41). Each 6-plex contained triplicate samples of dexamethasone-and DMSO-treated cells.…”

Section: Tmt Labeling Of Peptidesmentioning

confidence: 99%

Glucocorticoids Induce the Expansion of an Immature Human CFU-E Population

Ashley

Yan

Wang

et al. 2019

Preprint

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Despite effective clinical use, the mechanistic bases for the regulation of human erythropoiesis by glucocorticoids remain poorly understood. Here, we employed an erythroid culture system to differentiate primary human CD34 + cells isolated from control peripheral blood, cord blood and patients with Diamond Blackfan anemia (DBA), as model of an erythropoietic disorder treated with glucocorticoids. We found that the response to Dexamethasone is dependent on the developmental origin of the source of CD34 + cells, specifically increasing the expansion of CD34 + cells from peripheral blood but not cord blood. We also demonstrated that Dex treatment leads to the expansion of a novel immature colony-forming unit-erythroid (CFU-E) population in peripheral blood which is uniquely responsible for the proliferative effects observed during human adult erythropoiesis. Dex treatment of peripheral blood CFU-E cells also induced p57 Kip2 expression while patients with DBA resistant to steroids had altered p57 Kip2 expression that did not increase with Dex. Furthermore, shRNA knockdown of p57 Kip2 reduced proliferation and abrogated the effects of Dex. Finally, proteomics of Dex-treated CFU-E from peripheral blood and cord blood additionally revealed novel Dex targets in the erythroid system.Altogether, these results provide novel insights into the regulation of human erythropoiesis by glucocorticoids.3 Introduction:

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TMT Labeling for the Masses: A Robust and Cost-efficient, In-solution Labeling Approach

Abstract: Highlights • TMT labeling protocol with excellent intra-and interlaboratory reproducibility. • Complete in-solution labeling of peptides using 1/8 of recommended TMT quantities. • Demonstration of utility for deep-scale (phospho)proteome analysis.

Cited by 287 publications

References 28 publications

UbiFast, a rapid and deep-scale ubiquitylation profiling approach for biology and translational research

UbiFast, a rapid and deep-scale ubiquitylation profiling approach for biology and translational research

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

Glucocorticoids Induce the Expansion of an Immature Human CFU-E Population

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