E-resources
-
Zecha, Jana; Satpathy, Shankha; Kanashova, Tamara; Avanessian, Shayan C.; Kane, M. Harry; Clauser, Karl R.; Mertins, Philipp; Carr, Steven A.; Kuster, Bernhard
Molecular & cellular proteomics, 07/2019, Volume: 18, Issue: 7Journal Article
Isobaric labeling using tandem mass tags (TMTs) is increasingly applied for deep-scale proteomic studies in a multitude of organisms and addressing diverse research questions. The cost of labeling reagents represents a substantial proportion of the total expenses for conducting such experiments. Here, Zecha et al. present an economically optimized TMT labeling approach that reduces the quantity of required labeling reagent by a factor of eight and reproducibly achieves complete labeling. Display omitted 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. Isobaric stable isotope labeling using, for example, tandem mass tags (TMTs) is increasingly being applied for large-scale proteomic studies. Experiments focusing on proteoform analysis in drug time course or perturbation studies or in large patient cohorts greatly benefit from the reproducible quantification of single peptides across samples. However, such studies often require labeling of hundreds of micrograms of peptides such that the cost for labeling reagents represents a major contribution to the overall cost of an experiment. Here, we describe and evaluate a robust and cost-effective protocol for TMT labeling that reduces the quantity of required labeling reagent by a factor of eight and achieves complete labeling. Under- and overlabeling of peptides derived from complex digests of tissues and cell lines were systematically evaluated using peptide quantities of between 12.5 and 800 μg and TMT-to-peptide ratios (wt/wt) ranging from 8:1 to 1:2 at different TMT and peptide concentrations. When reaction volumes were reduced to maintain TMT and peptide concentrations of at least 10 mm and 2 g/l, respectively, TMT-to-peptide ratios as low as 1:1 (wt/wt) resulted in labeling efficiencies of > 99% and excellent intra- and interlaboratory reproducibility. The utility of the optimized protocol was further demonstrated in a deep-scale proteome and phosphoproteome analysis of patient-derived xenograft tumor tissue benchmarked against the labeling procedure recommended by the TMT vendor. Finally, we discuss the impact of labeling reaction parameters for N-hydroxysuccinimide ester-based chemistry and provide guidance on adopting efficient labeling protocols for different peptide quantities.
Shelf entry
Permalink
- URL:
Impact factor
Access to the JCR database is permitted only to users from Slovenia. Your current IP address is not on the list of IP addresses with access permission, and authentication with the relevant AAI accout is required.
Year | Impact factor | Edition | Category | Classification | ||||
---|---|---|---|---|---|---|---|---|
JCR | SNIP | JCR | SNIP | JCR | SNIP | JCR | SNIP |
Select the library membership card:
If the library membership card is not in the list,
add a new one.
DRS, in which the journal is indexed
Database name | Field | Year |
---|
Links to authors' personal bibliographies | Links to information on researchers in the SICRIS system |
---|
Source: Personal bibliographies
and: SICRIS
The material is available in full text. If you wish to order the material anyway, click the Continue button.