Low-Molecular-Weight Plasma Proteome Analysis Using Top-Down Mass Spectrometry.

Area of Science:

• Proteomics
• Biomarker Discovery
• Human Plasma Analysis

Background:

• Human plasma proteome contains valuable diagnostic information.
• Low molecular weight (LMW) proteome (<30 kDa) is a rich source of potential diagnostic biomarkers.
• Immunoaffinity depletion can remove high-abundance proteins but may affect LMW proteome analysis.

Purpose of the Study:

• To describe a protocol for top-down proteomic analysis of LMW proteoforms in human plasma.
• To identify and characterize LMW proteoforms without and with varying levels of high-abundance protein depletion.
• To establish a method for comprehensive LMW proteome profiling.

Main Methods:

• Fractionation of plasma samples by molecular weight using gel-eluted liquid fraction entrapment electrophoresis (GELFrEE).
• Nanocapillary-liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of GELFrEE fractions (<30 kDa).
• High-resolution mass spectrometry data processing using ProSightPC software.

Main Results:

• Identification and characterization of 442 LMW proteins and cleaved products across four human plasma sample types.
• Detection of LMW proteoforms with posttranslational modifications (PTMs) and single amino acid variations (SAAVs).
• Successful analysis without immunoaffinity depletion and with depletion of top 2, 6, and 7 high-abundance proteins.

Conclusions:

• The developed protocol enables comprehensive top-down proteomic analysis of the LMW proteome in human plasma.
• The identified LMW proteins, PTMs, and SAAVs represent a valuable resource for biomarker discovery.
• This method provides insights into the diagnostic potential of the low molecular weight human plasma proteome.