Enrichment of low molecular weight serum proteins using acetonitrile precipitation for mass spectrometry based proteomic analysis

Abstract

A rapid acetonitrile (ACN)-based extraction method has been developed that reproducibly depletes high abundance and high molecular weight proteins from serum prior to mass spectrometric analysis. A nanoflow liquid chromatography/tandem mass spectrometry (nano-LC/MS/MS) multiple reaction monitoring (MRM) method for 57 high to medium abundance serum proteins was used to characterise the ACN-depleted fraction after tryptic digestion. Of the 57 targeted proteins 29 were detected and albumin, the most abundant protein in serum and plasma, was identified as the 20th most abundant protein in the extract. The combination of ACN depletion and one-dimensional nano-LC/MS/MS enabled the detection of the low abundance serum protein, insulin-like growth factor-I (IGF-I), which has a serum concentration in the region of 100 ng/mL. One-dimensional sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the depleted serum showed no bands corresponding to proteins of molecular mass over 75 kDa after extraction, demonstrating the efficiency of the method for the depletion of high molecular weight proteins. Total protein analysis of the ACN extracts showed that approximately 99.6% of all protein is removed from the serum. The ACN-depletion strategy offers a viable alternative to the immunochemistry-based protein-depletion techniques commonly used for removing high abundance proteins from serum prior to MS-based proteomic analyses.

A proteomic approach combining MS and bioinformatic analysis for the detection and identification of biomarkers of administration of exogenous human growth hormone in humans

“…The 1500 most intense ions had an average %CV value of 37.7%, which could be attributed to a combination of experimental error and natural variances in endogenous serum protein concentrations between the individual males. Previous investigations using ACN depletion of serum proteins and a quantitative multiple reaction monitoring-based LC-MS/MS analysis demonstrated highly effective depletion of albumin and good reproducibility of low-molecular-weight protein enrichment [26]. The reproducibility of the m/z ion intensities indicates the ACN depletion method, combined with tryptic digestion and LC-MS analysis, and is suitable for biomarker discovery experiments.…”

“…Serum from rhGH-(n 5 21) and placebo-treated states (n 5 21) was extracted using an ACN depletion method developed in house [26]. ACN depletion is a reproducible and efficient technique for the removal of high-abundant and highmolecular-weight serum proteins (such as albumin), which complicate serum proteomic experiments.…”

Human POMC processing in vitro and in vivo revealed by quantitative peptidomics

“…EDTA was then removed and cells were directly lysed by vigorously pipetting cells with 80% acetonitrile (ACN, Pierce Cat# 51101), an organic solvent that is routinely used to precipitate larger proteins and protein complexes 53,73 . This lysate was then transferred to low protein-binding microcentrifuge tube (Sigma, Cat# Z666505), vortexed for 1 minute, and pelleted for 15 minutes at 12,000 g at 4˚C.…”

N-glycoproteomics: mass spectrometry-based glycosylation site annotation

“…The purifi cation effi ciency can, however, be enhanced by modifying the concentration of ion pairing reagents such as trifl uoroacetic acid, thereby reducing the overlap between glycopeptides and nonmodifi ed species (Mysling et al , 2010 ). However, secondary effects of the procedure have to be taken into account, as proteins (Kay et al , 2008 ) and peptides can be precipitated at increased ratios of organic solvent.…”