Comprehensive Analysis of Low-Molecular-Weight Human Plasma Proteome Using Top-Down Mass Spectrometry

Abstract

While human plasma serves as a great source for disease diagnosis, low-molecular-weight (LMW) proteome (<30 kDa) has been shown to contain a rich source of diagnostic biomarkers. Here we employ top-down mass spectrometry to analyze the LMW proteoforms present in four types of human plasma samples pooled from three healthy controls (HCs) without immunoaffinity depletion and with depletion of the top two, six, and seven high-abundance proteins. The LMW proteoforms were first fractionated based on molecular weight using gel-eluted liquid fraction entrapment electrophoresis (GELFrEE). Then, the GELFrEE fractions containing up to 30 kDa were subjected to nanocapillary-LC-MS/MS, and the high-resolution MS and MS/MS data were processed using ProSightPC 3.0. As a result, a total of 442 LMW proteins and cleaved products, including those with post-translational modifications and single amino acid variations, were identified. From additional comparative analysis of plasma samples without immunoaffinity depletion between HCs and colorectal cancer (CRC) patients via top-down approach, tens of LMW proteoforms, including platelet factor 4, were found to show >1.5-fold changes between the plasma samples of HCs and CRC patients, and six of the LMW proteins were verified by Western blot analysis.

Proteomic profiling of human plasma for cancer biomarker discovery

“…Recently, Cheon et al. performed a top‐down analysis of the low molecular weight (LMW) proteome in human plasma, and identified many LMW proteins, including platelet factor 4 that is a potential biomarker for colorectal cancer . Six of the LMW proteins were further validated by Western blot analysis.…”

Circulating MicroRNA Expression Levels Associated With Internet Gaming Disorder

“…To explore whether their protein expressions levels of the downstream target genes are different between the IGD and control groups, we selected 2 genes ( DUSP4 and PI15 ), which are predicted as downstream targets of all 3 miRNAs and additional 3 genes ( GABRB2, DPYSL2 , and CNR1 ) from those predicted for 2 miRNAs and performed western blot analysis with the plasma samples from 28 IGDs and 28 controls available for the experiment. We compared the expressions of the five targets between the IGD and control groups by measuring the band intensity and area as described elsewhere ( 29 ). Among them, the expression levels of DPYSL2 (28 IGDs and 28 controls, P = 0.0037) and GABBR2 (27 IGDs and 28 controls, P = 0.0052) were significantly higher in the IGD group (Figure 3 ).…”

“…We quantified the western blot results using the TotalLab 1D analysis software (Non-linear Dynamics, Newcastle upon Tyne, UK). Then, the densitometry ratio value was calculated by dividing the densitometry value of each sample as described elsewhere ( 29 ). As a control for normalization, a serum sample pooled from 46 IGD and control samples was used for every experiment.…”

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“…From preliminary proteomic analysis of HDL samples based on the protocol we previously reported [ 22 ], we selected five HDL-related proteins that were abundantly and reproducibly detected: apoA1, apoA2, apoC1, poC2, and apoC3. The proteins were measured and quantified as follows.…”