A method for the selective isolation and enrichment of carrier protein‐bound low‐molecular weight proteins and peptides in the blood

Abstract

The low molecular weight (LMW) region of the circulatory proteome, thought to contain a rich source of biomarkers, resides in vivo, in a complexed state with larger, highly abundant resident proteins. Consequently, serum fractionation approaches that deplete the high-abundance proteins under native conditions will remove much of the LMW proteome. We describe a new strategy to systematically collect, isolate and enrich the LMW molecules that would be otherwise eliminated during the depletion of high-abundance circulatory proteins based on continuous elution electrophoresis. We employ strong denaturing conditions to disrupt association with the high-abundance carrier proteins followed by fractionation and removal of SDS. Under denaturation, the LMW molecules were effectively stripped from the highly abundant carrier proteins. We then removed the SDS by ion exchange matrix sequestration and concentrated the fractions. The outcome is a series of SDS-free fractions of LMW molecules. The isolated fractions were then analyzed by enzymatic digestion followed by LC-MS/MS analysis. The yield of multiple peptide hits as well as the total number of identifications significantly increased (50%) compared to unfractionated serum. The method yielded a 30% higher number of low-abundance serum proteins compared to direct sequencing of unfractionated serum.

Automated analysis of mouse serum peptidome using restricted access media and nanoliquid chromatography–tandem mass spectrometry

“…Accordingly, three LC-MS/MS repeats provide comprehensive identification of the proteins present in the tryptic digested fractions. These results are remarkably better than those reported by Camerini et al which ran a total of eight repeats: three repeats identified about 70% of the proteins, whereas seven repeats were required to identified 98% of the proteins [16]. In the followon experiments, we ran three LC-MS/MS repeats for each fraction of each sample/experiment set.…”

“…Ultrafiltration with 10-30 kDa filters has been used frequently for selective removal of high abundance, larger proteins [11][12][13][14] though some leakage of high molecular weight molecules and great variance in cut-off accuracy has been noted [15]. Electrophoretic methods [16] and size exclusion chromatography [17] also have shown promise in this regard but, similar to ultrafiltration, the small sample load and low throughput have limited their utility in biomarker discovery applications.…”

Smart Hydrogel Particles: Biomarker Harvesting: One-Step Affinity Purification, Size Exclusion, and Protection against Degradation

“…The particles were heated in SDS sample buffer for 5 minutes at 100 °C and loaded on a 4-20 % Tris Glicine gel (Invitrogen). Bands below 30 kDa were cut and ingel trypsin digestion was performed 11 . The resulting peptide fragments were analyzed by online liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI MS) using LTQ-Orbitrap mass spectrometer (Thermo Fisher).…”

Recent advances of mesoporous materials in sample preparation

“…However, due to the complexity and high dynamic range of endogenous peptides and the interference from high concentrations of proteins, salts and lipids in biological samples, the ability to extract peptides/small proteins from the complex biological samples as well as enhancing the sensitivity toward peptides/small proteins at low abundance, remains a great challenging task before MS analysis. Thus, various methods (ultrafiltration [62], solid-phases extraction [63], selective electrophoresis and continuous elution electrophoresis [64], organic solvent precipitation [65]) have been developed to isolate peptides/small proteins before MS analysis. Centrifugal ultrafiltration with accurate MW cutoff is one widely used method for peptide enrichment based on size-exclusion effect.…”