Top-Down Mass Spectrometry on Tissue Extracts and Biofluids with Isoelectric Focusing and Superficially Porous Silica Liquid Chromatography

Abstract

Top-down mass spectrometry (MS) has emerged as a powerful complement to peptide-based proteomics. Despite advancements, the field has had limited application to clinical proteomics investigations due to the complexity and poor dynamic range of chromatography used to separate intact proteins from tissue and biofluids. To address these limitations, we developed a two-dimensional (2D) chromatography platform that includes isoelectric focusing (IEF) through immobilized pH gradient and superficially porous liquid chromatography (SPLC). Analysis of standard proteins demonstrates compatibility of IEF-SPLC processing and high resolving-power MS analysis with results showing ~7.0 femtomole detection limits and linear spectral response for proteins fractionated over ~4 log sample loads. For proteins from heart myofibrils and cerebrospinal fluid (CSF), compared to one-dimensional SPLC-MS, the 2D IEF-SPLC-MS platform resulted in a 5-6× increase in the number of unique monoisotopic masses observed <30 kDa and an ~4× improved mass range enabling the observation of proteins >200 kDa. In the heart myofibrils, common protein proteoforms observed were associated with phosphorylation of contractile proteins with results showing that quantitative evaluation of their PTM stoichiometry was possible despite differentially modified forms being fractionated into separate pI compartments. In CSF, diverse protein mutations and PTM classes were also observed, including differentially glycosylated protein forms separated to different pI. Results also demonstrate that by the generation of IEF-SPLC protein libraries by fraction collection, the platform enables prospective protein identification and proteoform analysis investigations by complementary top-down and bottom-up strategies. Overall, the 2D platform presented may provide the speed, dynamic range, and detection limits necessary for routine characterization of proteoform-based biomarkers from biofluids and tissues.

Top-Down Proteomics of Large Proteins up to 223 kDa Enabled by Serial Size Exclusion Chromatography Strategy

“…21,37,38 Zhang et al employed isoelectric focusing and superficially porous silica LC for the separation of mouse cardiac tissue extract, and were able to detect myosin heavy chain variants. 39 However, the coverage of the cardiac proteome was limited. The sSEC method developed in this study enabled robust and high-resolution size-based separation of proteins over a broad MW range.…”

An off-line high pH reversed-phase fractionation and nano-liquid chromatography–mass spectrometry method for global proteomic profiling of cell lines

“…First-dimension separation methods include isoelectric focusing electrophoresis (IEF) [1][2][3][4][5], on-line multiple-junction capillary isoelectric focusing fractionator (OMJ-CIEF) [6], one-dimensional and two-dimensional gel electrophoresis [7][8][9], IEF with superficially porous liquid chromatography (IEF-SPLC) [10], off-line and on-line high performance liquid chromatographic (HPLC) separation [11][12][13], two-dimensional (2D)-LC systems with online fractionation of proteins into a series of small trapping columns [14], 2D-LC-UV/MS analysis [15] and three-dimensional approaches [16,17]. All these methods provide important tools for identifying and quantifying proteomic difference between multiple biological conditions.…”

“…Collected fractions can be further processed, http://dx.doi.org/10.1016/j.jchromb.2014. 10.031 1570-0232/© 2014 Elsevier B.V. All rights reserved. concentrated, enriched or chemically modified prior to analysis in the second capillary or nano-LC-MS dimension.…”

Continuous Elution Proteoform Identification of Myelin Basic Protein by Superficially Porous Reversed-Phase Liquid Chromatography and Fourier Transform Mass Spectrometry

“…TDMS typically employs LC, intact mass analysis, and gas-phase fragmentation (e.g., tandem mass spectrometry, MS/MS) to characterize protein’s sequences, localize the positions of their PTMs, and quantify related proteoform ratios. 21–23 While applied in comprehensive proteomics environments that identify thousands of proteins in samples, 24 TDMS has proven especially valuable for in-depth proteoform investigations (e.g., histones, glycoproteins, cardiac proteins, and virulence factors 22,25–30 ) that are aided by the high resolving power and mass accuracy afforded by Fourier transform mass spectrometry (FTMS), 31,32 as well as offline and online data-dependent (DD) fragmentation, diverse 1D and 2D LC techniques, and specialized bioinformatics tools. 20,35,36 Herein, we highlight a new workflow that combines superficially porous reversed-phase liquid chromatography (SPLC) and fragmentation by data-independent acquisition (DIA) on a FTMS with a custom bioinformatics workflow 31,35 for the rapid interrogation of the MBP proteoform landscape.…”