Multidimensional separations in top–down proteomics

Abstract

Top-down proteomics (TDP) identifies, quantifies, and characterises proteins at the intact proteoform level in complex biological samples to understand proteoform function and cellular mechanisms. However, analysing complex biological samples using TDP is still challenging due to high sample complexity and wide dynamic range. Highresolution separation methods are often applied prior to mass spectrometry (MS) analysis to decrease sample complexity and increase proteomics throughput. These separation methods, however, may not be efficient enough to characterise low abundance intact proteoforms in complex samples. As such, multidimensional separation techniques (combination of two or more separation methods with high orthogonality) have been developed and applied that demonstrate improved separation resolution and more comprehensive identification in TDP. A suite of multidimensional separation methods that couple various types of liquid chromatography (LC), capillary electrophoresis (CE), and/or gel electrophoresis-based separation approaches have been developed and applied in TDP to analyse complex biological samples. Here, we reviewed multidimensional separation strategies employed for TDP, summarised current applications, and discussed the gaps that may be addressed in the future.

Characterizing Age-related Changes in Intact Mitochondrial Proteoforms in Murine Hearts using Quantitative Top-Down Proteomics

“…For example, other separation techniques such as capillary electrophoresis that have higher sensitivity and separation e ciency compared with LC methods have been implemented for top-down proteomics [71][72][73][74]. Additionally, multidimensional separation of intact proteins has been shown to improve separation e ciency and allow for the characterization of more intact proteoforms in complex samples [21,[75][76][77][78][79][80][81]. However, label-free quantitation techniques as used here are challenging to implement with multidimensional separation methods.…”

Influence of Different Sample Preparation Approaches on Proteoform Identification by Top-Down Proteomics

“…For comprehensive TDP studies, multidimensional separation schemes are typically utilized to increase the number of identi cations. 13 To this end, the effects of an LC-and a gel-based separation approach on the identi cation of proteoforms were investigated using a recently developed two-dimensional low/low pH reversed-phase LC separation scheme 29 and the GELFrEE system, 24 respectively (Supplementary Results).…”

“…3 Despite signi cant advances made in the recent past, [4][5][6][7][8][9][10] the proteome-wide identi cation of proteoforms by TDP is still hampered by several challenges at all stages of the analysis, i.e., sample preparation, proteoform separation, mass spectrometric analysis, and database search. [11][12][13] These challenges become more signi cant with the increasing size of proteoforms. For example, the sensitivity in electrospray-MS inherently decreases with increasing proteoform size since the intensity is split into multiple charge states and isotopologues.…”

Top-down Proteomics Analysis of Picogram-level Complex Samples using Spray-Capillary-Based Capillary Electrophoresis Mass Spectrometry

“…Optimization of this intact protein TMT labeling platform for single cell analysis for multiplexed top-down proteomics would increase the injected mass and improve proteoform identification. Additionally, multidimensional separations have led to improved proteoform identification in top-down proteomics [57][58][59] . Coupling nanoLC with the spray-capillary CE-MS may also improve proteoform identification of limited mass samples and single cells.…”