Small-protein Enrichment Assay Enables the Rapid, Unbiased Analysis of Over 100 Low Abundance Factors from Human Plasma

Abstract

Small proteins in plasma regulate diverse processes from metabolism to immune functions and are difficult to study, because of the large dynamic range of protein abundance. To facilitate their detection, we developed a method using protein dissociation, precipitation and size exclusion chromatography called SPEA. Herein, we demonstrate SPEA is capable of reproducible characterization of known small proteins and identification of novel factors such as C5ORF46. When applied to study humans undergoing intermittent fasting, novel changes in iron regulation were observed.

Multi-omics Analysis of the Intermittent Fasting Response in Mice Identifies an Unexpected Role for HNF4α

“…One fasting-responsive signaling pathway known to be upstream of IGF-1 secretion from the liver is AMP-activated protein kinase (AMPK) (Chen et al, 2016), which may be a key mediator of this EODF phenotype. IGF-1 is also known to be sensitive to proteolytic degradation in plasma (Harney et al, 2019a;Yamamoto and Murphy, 1994), and these proteases may be further activated due to either the decreased abundance of protease inhibitors, such as a1-antitrypsin, or the increased plasma carboxypeptidase Q protein abundance we observed after EODF. Regardless of the cause, the decreased IGF-1 level we observe in plasma would correlate with the increased lifespan in these animals.…”

Aqueous Fibronectin Correlates With Severity of Macular Edema and Visual Acuity in Patients With Branch Retinal Vein Occlusion: A Proteome Study

“…49 Fragmentation efficiency of tryptic peptides remains a limiting factor in identification of lowly abundant proteins. 48 Furthermore, low-abundant proteins with low molecular weight, for example interleukin-8, yield very few tryptic peptides, which makes them complicated to identify with mass spectrometry. 50 The sample material is another limitation of our study.…”

“…Sample complexity with high dynamic ranges of protein abundances makes detection of low-abundant proteins difficult as protein abundances may stretch over 13 orders of magnitude depending on the material under study. 48 Low abundance proteins tend to yield larger spectral count variation resulting in underestimation of differences in expression level. 49 Fragmentation efficiency of tryptic peptides remains a limiting factor in identification of lowly abundant proteins.…”

Complementarity of Different SDS‐PAGE Gel Staining Methods for the Identification of Short Open Reading Frame‐Encoded Peptides

“…Therefore, special approaches have to be used, for example, enrichment of small or depletion of larger proteins. A number of methodologies have been described, for example the use of molecular weight cut‐off filters, [ 10 ] the enrichment of small proteins by the GELFrEE‐system [ 11 ] the application of size exclusion chromatography [ 12 ] or of solid phase extraction [ 13 ] and the depletion of high molecular weight proteins by acetonitrile‐precipitation [ 14 ] or acetic acid precipitation. [ 15 ] However, these methodologies favor and disfavor particular proteins and therefore the development of other potentially complementary methodologies for the analysis of small proteins is necessary.…”