Deep Intact Proteoform Characterization in Human Cell Lysate using High-pH and Low-pH Reversed-Phase Liquid Chromatography

Post-translational modifications (PTMs) play critical roles in biological processes and have significant effects on the structures and dynamics of proteins. Top-down proteomics methods were developed for and applied to the study of intact proteins and their PTMs in human samples. However, the large dynamic range and complexity of human samples makes the study of human proteins challenging. To address these challenges, we developed a 2D pH RP/RPLC-MS/MS technique that fuses high-resolution separation and intact protein characterization to study the human proteins in HeLa cell lysate. Our results provide a deep coverage of soluble proteins in human cancer cells. Compared to 225 proteoforms from 124 proteins identified when 1D separation was used, 2778 proteoforms from 628 proteins were detected and characterized using our 2D separation method. Many proteoforms with critically functional PTMs including phosphorylation were characterized. Additionally, we present the first detection of intact human GcvH proteoforms with rare modifications such as octanoylation and lipoylation. Overall, the increase in the number of proteoforms identified using 2DLC separation is largely due to the reduction in sample complexity through improved separation resolution, which enables the detection of low abundance PTM modified proteoforms. We demonstrate here that 2D pH RP/RPLC is an effective technique to analyze complex protein samples using top-down proteomics

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

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

Identification and Quantification of Proteoforms by Mass Spectrometry

A proteoform is a defined form of a protein derived from a given gene with a specific amino acid sequence and localized post-translational modifications. In top-down proteomic analyses, proteoforms are identified and quantified through mass spectrometric analysis of intact proteins. Recent technological developments have enabled comprehensive proteoform analyses in complex samples, and an increasing number of laboratories are adopting top-down proteomic workflows. In this review, we outline some recent advances and discuss current challenges and future directions for the field

Developments of high-throughput quantitative top-down proteomics

The LC-MS techniques are commonly used to analyze intact proteoforms for top-down proteomics. To deepen the coverage of the intact human proteome, multidimensional separations are often applied prior to the MS analysis. However, the most common quantitative top-down approach, label-free quantification, cannot be applied to multidimensional separations because proteins that elute in multiple fractions cannot be quantified. The focus of this dissertation is the development of high-throughput quantitative top-down proteomics techniques for the analysis of complex biological samples (e.g., bacteria and human cell lysate) using isobaric chemical tag labeling for application to multidimensional separation and quantitative top-down proteomics. To improve the intact proteome coverage of human protein samples, we applied a 2D pH RP/RPLC-MS platform to characterize intact human proteoforms. The high resolution of RPLC and orthogonality between high-pH and low-pH RPLC separations provide an aerial view of the intact human proteome. Our results demonstrated that the 2DLC platform dramatically improves the identification of intact proteins and proteoforms, and largely enhances the detection of low abundant proteoforms and PTMs. The isobaric chemical tag labeling quantification has been widely applied in bottom-up proteomics. However, protein-level labeling is challenging due to the tendency of intact proteins to aggregate under labeling conditions. We developed a filter-SEC approach to enrich low MW proteins from the complex sample, these proteins maintain solubility in protein-level TMT labeling for quantitative top-down MS analysis. Once the protein aggregation issue had been addressed, however, we found that protein-level TMT labeling under standard labeling conditions resulted in unwanted side products (underlabeled or overlabeled species). This issue is more serious for intact proteoform labeling because a higher number of residues are potentially labeled when compared with shorter peptides. We comprehensively optimized and evaluated the protein-level TMT labeling conditions including TMT to protein ratio, final pH (quenching solution concentration), reaction concentration (starting sample volume), reaction time, and reaction buffers. After optimization of parameters, we found that 85% of the proteoforms were completely labeled after the reaction; this improvement in labeling efficiency allows protein-level isobaric chemical tag labeling to be used for real applications. Overall, our results demonstrate the potential of quantitative top-down proteomics using TMT and multidimensional separation. High throughput quantitative top-down proteomics techniques using protein-level TMT labeling hold great potential for the relative quantification of intact proteoforms. We hope that our work can push the isobaric chemical tag labeling quantification for intact proteoforms into practical applications

Development of an Online 2D Ultrahigh-Pressure Nano-LC System for High-pH and Low-pH Reversed Phase Separation in Top-Down Proteomics

The development of novel high-resolution separation techniques is crucial for advancing the complex sample analysis necessary for high-throughput top-down proteomics. Recently, our group developed an offline 2D high-pH RPLC/low-pH RPLC separation method and demonstrated good orthogonality between these two RPLC formats. Specifically, ultrahigh-pressure long capillary column RPLC separation has been applied as the second dimensional low-pH RPLC separation for the improvement of separation resolution. To further improve the throughput and sensitivity of the offline approach, we developed an online 2D ultrahigh-pressure nano-LC system for high-pH and low-pH RPLC separations in top-down proteomics. An online microtrap column with a dilution setup was used to collect eluted proteins from the first dimension high-pH separation and inject the fractions for ultrahigh-pressure long capillary column low-pH RPLC separation in the second dimension. This automatic platform enables the characterization of 1000+ intact proteoforms from 5 μg of intact E. coli cell lysate in 10 online-collected fractions. Here, we have demonstrated that our online 2D pH RP/RPLC system coupled with top-down proteomics holds the potential for deep proteome characterization of mass-limited samples because it allows the identification of hundreds of intact proteoforms from complex biological samples at low microgram sample amounts