Quantitative Top-Down Proteomics in Complex Samples Using Protein-Level Tandem Mass Tag Labeling

Abstract

Labeling approaches using isobaric chemical tags (e.g., isobaric tagging for relative and absolute quantification, iTRAQ and tandem mass tag, TMT) have been widely applied for the quantification of peptides and proteins in bottom-up MS. However, until recently, successful applications of these approaches to top-down proteomics have been limited because proteins tend to precipitate and “crash” out of solution during TMT labeling of complex samples making the quantification of such samples difficult. In this study, we report a top-down TMT MS platform for confidently identifying and quantifying low molecular weight intact proteoforms in complex biological samples. To reduce the sample complexity and remove large proteins from complex samples, we developed a filter-SEC technique that combines a molecular weight cutoff filtration step with high-performance size exclusion chromatography (SEC) separation. No protein precipitation was observed in filtered samples under the intact protein-level TMT labeling conditions. The proposed top-down TMT MS platform enables high-throughput analysis of intact proteoforms, allowing for the identification and quantification of hundreds of intact proteoforms from Escherichia coli cell lysates. To our knowledge, this represents the first high-throughput TMT labeling-based, quantitative, top-down MS analysis suitable for complex biological samples.

Quantitative Top-Down Proteomics by Isobaric Labeling with Thiol-Directed Tandem Mass Tags

“…39 To circumvent this side reaction, higher amounts of organic solvents in the reaction mixture are applied, which can lead to protein precipitation, particularly for larger proteins. 35 Therefore, our workflow obviates the need for extensive depletion of larger proteoforms prior to labeling as suggested before. 35 Insulin chain A (2.4 kDa, 4 Cys) and chain B (3.4 kDa, 2 Cys), α-lactalbumin (14.2 kDa, 8 Cys), lysozyme (14.3 kDa, 8 Cys), and β-lactoglobulin (18.4 kDa, 5 Cys) were used as standard proteins to test this hypothesis and to evaluate the specificity of the labeling reaction with iodoTMTzero.…”

“…35 Therefore, our workflow obviates the need for extensive depletion of larger proteoforms prior to labeling as suggested before. 35 Insulin chain A (2.4 kDa, 4 Cys) and chain B (3.4 kDa, 2 Cys), α-lactalbumin (14.2 kDa, 8 Cys), lysozyme (14.3 kDa, 8 Cys), and β-lactoglobulin (18.4 kDa, 5 Cys) were used as standard proteins to test this hypothesis and to evaluate the specificity of the labeling reaction with iodoTMTzero. The labeled proteins showed quantitative derivatization of the target sites, with a low degree of under-or overlabeling (Figure S3).…”

“…We therefore assumed lower amounts of over-and underlabeling using cysteine-reactive iodoTMT compared to amino-directed TMT reagents, which have been described to cause an increase in sample complexity. 34,35 Another advantage is the relative stability of iodoTMT reagents in aqueous solutions. Amino-directed TMT reagents are activated N-hydroxysuccinimide esters, which can undergo hydrolysis, especially under basic conditions.…”

“…34 This principle was recently extended by the development of a labeling platform for complex samples, which allowed quantification of over 400 E. coli proteoforms from two liquid chromatography (LC)−MS runs. 35 To prevent protein precipitation during labeling, a multistep depletion of larger proteoforms (>30 kDa) encompassing two molecular weight cutoff (MWCO) steps and a separation via size exclusion chromatography (SEC) were part of this workflow. These steps are performed prior to labeling, which bears the potential to introduce sampling bias.…”

“…Additionally, labeling with amino-reactive TMT can introduce significant sample heterogeneity at the intact protein level, especially for larger proteoforms. 34,35 Here, we describe the development of a labeling strategy for TDP targeting thiol groups of cysteine residues with iodoTMTsixplex reagents. While this method inherently excludes the quantification of cysteine-free proteoforms, it provides the opportunity of sixplexing and the implementation of multidimensional separation schemes to increase proteome coverage.…”