Top-Down Proteomics of Large Proteins up to 223 kDa Enabled by Serial Size Exclusion Chromatography Strategy
Open accessAbstract
Mass spectrometry (MS)-based top-down proteomics is a powerful method for the comprehensive analysis of proteoforms that arise from genetic variations and post-translational modifications (PTMs). However, top-down MS analysis of high molecular weight (MW) proteins remains challenging mainly due to the exponential decay of signal-to-noise ratio with increasing MW. Size exclusion chromatography (SEC) is a favored method for size-based separation of biomacromolecules but typically suffers from low resolution. Herein, we developed a serial size exclusion chromatography (sSEC) strategy to enable high-resolution size-based fractionation of intact proteins (10-223 kDa) from complex protein mixtures. The sSEC fractions could be further separated by reverse phase chromatography (RPC) coupled online with high-resolution MS. We have shown that two-dimensional (2D) sSEC-RPC allowed for the identification of 4044 more unique proteoforms and a 15-fold increase in the detection of proteins above 60 kDa, compared to one-dimensional (1D) RPC. Notably, effective sSEC-RPC separation of proteins significantly enhanced the detection of high MW proteins up to 223 kDa and also revealed low abundance proteoforms that are post-translationally modified. This sSEC method is MS-friendly, robust, and reproducible and, thus, can be applied to both high-efficiency protein purification and large-scale proteomics analysis of cell or tissue lysate for enhanced proteome coverage, particularly for low abundance and high MW proteoforms.
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Single-dimension RPLC can identify only ~100 intact proteoforms with limited coverage of larger species (>30 kDa).
“Typically a single-dimension of RPLC can only enable the identification of ~100 intact proteoforms with limited coverage of larger species (>30 kDa) [69].”
From Top-down proteomics: challenges, innovations, and applications in basic and clinical research (2020) · cites this paper as [69]
sSEC achieved high-resolution size-based separation of cardiac sarcomere proteins and, when used for offline fractionation before RPLC-MS, enabled detection of ~4,000 unique proteoforms up to 223 kDa.
“sSEC was used to achieve high-resolution size-based separation of cardiac sarcomere proteins and, when used as for offline fractionation before RPLC-MS, enabled the detection of ~4,000 unique proteoforms up to 223 kDa [69].”
From Top-down proteomics: challenges, innovations, and applications in basic and clinical research (2020) · cites this paper as [69]
q-TOF mass spectrometers are a common alternative, robust, affordable, and capable of analyzing relatively large protein species.
“Quadrupole time-of-flight (q-TOF) mass spectrometers are a common alternative instrument platform, as they are robust, affordable, and capable of analyzing relatively large protein species [69,97].”
From Top-down proteomics: challenges, innovations, and applications in basic and clinical research (2020) · cites this paper as [69]
A recently developed 2DLC method coupling serial size-exclusion chromatography with reversed-phase chromatography enabled the detection and identification of larger heart proteins, including cMyBP-C and MHC, but required much larger amounts of tissue than the HCM septal myectomy tissues obtained.
“Although we have recently developed a 2DLC method coupling serial size-exclusion chromatography with reversed-phase chromatography that enabled the detection and identification of larger heart proteins, including cMyBP-C and MHC (48, 49), it required much larger amount of tissues than the HCM septal myectomy tissues we obtained here.”
From Distinct hypertrophic cardiomyopathy genotypes result in convergent sarcomeric proteoform profiles revealed by top-down proteomics (2020) · cites this paper as [48]
Size-based protein fractionation is essential to separate the larger proteins from the smaller ones for top-down MS analysis of large proteins.
“Size-based protein fractionation is essential to separate the larger proteins from the smaller ones for the top-down MS analysis of large proteins (47, 48).”
From Distinct hypertrophic cardiomyopathy genotypes result in convergent sarcomeric proteoform profiles revealed by top-down proteomics (2020) · cites this paper as [48]
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Self-citation
- Authors
- Cai, W., Tucholski, T., Chen, B., Alpert, A. J., McIlwain, S., Kohmoto, T., Jin, S., & Ge, Y.
- Affiliations
- University of Wisconsin–Madison (US)
- Corresponding author
- Ge, Ying
- Venue
- Analytical Chemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2017-05-02
- Volume
- 89
- Issue
- 10
- Pages
- 5467-5475
- ISSN
- 0003-2700 (ISSN-L)
- 1520-6882
- PMC
- PMC5511113
- OpenAlex
- W2606061971
- Cited by
- 135
- References
- 48
- Citations by year
- 2026: 11 · 2025: 7 · 2024: 11 · 2023: 16 · 2022: 12 · 2021: 15 · 2020: 16 · 2019: 25 · 2018: 13 · 2017: 8 · 2015: 1
- FWCI
- 8.36
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Analytical Chemistry (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Center for Advancing Translational Sciences (UL1TR000427)
- National Cancer Institute (P30 CA014520)
- National Heart, Lung, and Blood Institute (R01HL096971, R01HL109810)
- Office of the Director (S10OD018475)
- National Institute of General Medical Sciences (R01GM117058)
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
- Keywords
- top-down proteomics, high molecular weight proteins, proteoforms, post-translational modification, reverse-phase chromatography, signal-to-noise ratio, proteome coverage
- MeSH terms
- Chromatography, Gel, Chromatography, High Pressure Liquid, Humans, Molecular Weight, Myocardium, Proteins, Proteomics, Creatine Kinase, MM Form, Tandem Mass Spectrometry, Chromatography, Reverse-Phase
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