Orbitrap Mass Spectrometry and High-Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) Enable the in-Depth Analysis of Human Serum Proteoforms.
J Proteome Res
; 22(11): 3418-3426, 2023 11 03.
Article
em En
| MEDLINE
| ID: mdl-37774690
ABSTRACT
Blood serum and plasma are arguably the most commonly analyzed clinical samples, with dozens of proteins serving as validated biomarkers for various human diseases. Top-down proteomics may provide additional insights into disease etiopathogenesis since this approach focuses on protein forms, or proteoforms, originally circulating in blood, potentially providing access to information about relevant post-translational modifications, truncations, single amino acid substitutions, and many other sources of protein variation. However, the vast majority of proteomic studies on serum and plasma are carried out using peptide-centric, bottom-up approaches that cannot recapitulate the original proteoform content of samples. Clinical laboratories have been slow to adopt top-down analysis, also due to higher sample handling requirements. In this study, we describe a straightforward protocol for intact proteoform sample preparation based on the depletion of albumin and immunoglobulins, followed by simplified protein fractionation via polyacrylamide gel electrophoresis. After molecular weight-based fractionation, we supplemented the traditional liquid chromatography-tandem mass spectrometry (LC-MS2) data acquisition with high-field asymmetric waveform ion mobility spectrometry (FAIMS) to further simplify serum proteoform mixtures. This LC-FAIMS-MS2 method led to the identification of over 1000 serum proteoforms < 30 kDa, outperforming traditional LC-MS2 data acquisition and more than doubling the number of proteoforms identified in previous studies.
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Texto completo:
1
Bases de dados:
MEDLINE
Assunto principal:
Soro
/
Espectrometria de Mobilidade Iônica
Tipo de estudo:
Guideline
Limite:
Humans
Idioma:
En
Revista:
J Proteome Res
Assunto da revista:
BIOQUIMICA
Ano de publicação:
2023
Tipo de documento:
Article
País de afiliação:
Estados Unidos