A Quantitative Study of the Effects of Chaotropic Agents, Surfactants, and Solvents on the Digestion Efficiency of Human Plasma Proteins by Trypsin

Abstract

Plasma biomarkers studies are based on the differential expression of proteins between different treatment groups or between diseased and control populations. Most mass spectrometry-based methods of protein quantitation, however, are based on the detection and quantitation of peptides, not intact proteins. For peptide-based protein quantitation to be accurate, the digestion protocols used in proteomic analyses must be both efficient and reproducible. There have been very few studies, however, where plasma denaturation/digestion protocols have been compared using absolute quantitation methods. In this paper, 14 combinations of heat, solvent [acetonitrile, methanol, trifluoroethanol], chaotropic agents [guanidine hydrochloride, urea], and surfactants [sodium dodecyl sulfate (SDS) and sodium deoxycholate (DOC)] were compared with respect to their effectiveness in improving subsequent tryptic digestion. These digestion protocols were evaluated by quantitating the production of proteotypic tryptic peptides from 45 moderate- to high-abundance plasma proteins, using tandem mass spectrometry in multiple reaction monitoring mode, with a mixture of stable-isotope labeled analogues of these proteotypic peptides as internal standards. When the digestion efficiencies of these 14 methods were compared, we found that both of the surfactants (SDS and DOC) produced an increase in the overall yield of tryptic peptides from these 45 proteins, when compared to the more commonly used urea protocol. SDS, however, can be a serious interference for subsequent mass spectrometry. DOC, on the other hand, can be easily removed from the samples by acid precipitation. Examining the results of a reproducibility study, done with 5 replicate digestions, DOC and SDS with a 9 h digestion time produced the highest average digestion efficiencies (~80%), with the highest average reproducibility (<5% error, defined as the relative deviation from the mean value). However, because of potential interferences resulting from the use of SDS, we recommend DOC with a 9 h digestion procedure as the optimum protocol.

Tracheal Tissue Engineering

“…This approach utilizes decellularized human tracheas prepared via a detergent enzymatic method with sodium deoxycholate. In addition to dire supply limitations, sodium deoxycholate is harsh ionic detergent known to denature proteins both in vitro and in vivo [180,181,182,183,184].…”

“…Sodium deoxycholate, the most commonly employed detergent, is known to denature proteins both in vitro and in vivo [180,181,182,183,184]. More than simply removing cellular antigens and debris, ECM grafts processed in this way may not retain the growth factors, proteoglycans, and other matrix proteins in a native configuration capable of providing cues to infiltrating host cells, potentially degrading performance and outcomes.…”

“…This in turn allows more detergents to bind, known as cooperative binding, which can completely denature the higher order structure of the protein and effectively destroy it's biological function. For example, sodium deoxycholate, the detergent employed clinically, is often used to denature proteins prior to enzymatic degradation for proteomic analysis [180]. Ionic detergent monomer affinity also makes it harder to remove residual detergent from a decellularized tissue after processing.…”

Prefractionation and separation by C8 stationary phase: Effective strategies for integral membrane proteins analysis

“…However, the analysis of IMPs presents great challenges due to the hydrophobic nature [4][5][6][7]. Recently, much attention has been paid to the solubilization of IMPs, by using chaotropes [8][9][10], detergents [9][10][11][12][13][14][15][16][17], organic solvents [9,16,[18][19][20], organic acids [21][22][23] and ionic liquid [24]. Besides, the separation of hydrophobic peptides with high recovery also plays a crucial role in IMP identification.…”

Urea free and more efficient sample preparation method for mass spectrometry based protein identification via combining the formic acid-assisted chemical cleavage and trypsin digestion

“…For the favorable MS based protein identification, protein sample preparation [2,3] must be performed firstly using enzymes [4] or chemicals that cleave proteins at some amino acid (AA) sites for obtaining peptides. As the pivotal process, in the conventional urea-assisted protein sample preparation method, detergents [5][6][7] routinely used to denature proteins, such as urea and guanidine hydrochloride will disturb MS analysis to some extent. For instance, they will interfere with the whole process of matrix-assisted laser introduced recently to replace urea.…”