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.
Keywords: isobaric labeling; liquid chromatography; mass spectrometry; quantitative proteomics; tandem mass tags (TMT); top-down proteomics.