Detection and Identification of Sub-nanogram Levels of Protein in a NanoLC-Trypsin-MS System

Abstract

Proteomic workflows involving liquid-based protein separations are an alternative to gel-based protein analysis, however the trypsin digestion procedure is usually difficult to implement, particularly when processing low abundance proteins from capillary column effluent. To convert the protein to peptides for the purpose of identification, current protocols require several sample handling steps, and sample losses become an issue. In this study, we present an improved system that conducts reversed-phase protein chromatography and rapid on-line tryptic digestion requiring sub-nanogram quantities of protein. This system employs a novel mirror-gradient concept that allows for dynamic titration of the column effluent to create optimal conditions for real-time tryptic digestion. The purpose behind this development was to improve the limits of detection of the online concept, to support flow-based alternatives to gel-based proteomics and to simplify the characterization of low abundance proteins. Using test mixtures of proteins, we show that peptide mass fingerprinting with high sequence representation can be easily achieved at the 20 fmol level, with detection limits down to 5 fmol (85 pg myoglobin). Limits of identification using standard data-dependent MS/MS experiments are as low as 10 fmol. These results suggest that the nanoLC-trypsin-MS/MS system could represent an alternative to the conventional "1D-gel to MS" proteomic strategy.

Pressurized Pepsin Digestion in Proteomics

“…bottom-up) LC-MS analyses after digestion into an integrated platform have been attempted with varying degrees of success. Many approaches have been developed, and most of them use an integrated on-column microreactor based on some form of enzyme immobilization for the rapid digestion of protein fractions into peptides (11,45,46). Although many of these approaches were successful in digesting proteins on line, their application to demanding proteomics experiments proved to be limited because of rapid inactivation of the immobilized enzyme presumably by endogenous proteases, mobile phase additives and impurities, organic solvents, endogenous inhibitors, and other factors (47).…”

High throughput tryptic digestion via poly (acrylamide-co-methylenebisacrylamide) monolith based immobilized enzyme reactor

“…It could be also connected with related components directly for on-line digestion [9,10]. Nowadays, a variety of IMERs have been developed with different supporting materials, e.g., membranes [11][12][13], particles [14][15][16], monoliths and so on. Among them, the monolith based IMER [17][18][19][20][21] has drawn great attention due to its high capacity for enzymes, fast and simple preparation, low back-pressure, biological inertia and mechanical stability, rendering greatly enhanced digestion efficacy.…”

Recent advances on multidimensional liquid chromatography–mass spectrometry for proteomics: From qualitative to quantitative analysis—A review

“…However, most of protein-level prefractionations were performed offline which might result in the decreased sensitivity and analytical throughput. Recently, Slysz and Schriemer [83,84] proposed an approach to achieve protein separation by RPLC, on-line digestion by IMER and identification by peptide mass fingerprinting (PMF) search. High sequence coverage could be easily achieved at the 20 fmol level, with the limit of detection (LOD) down to 5 fmol for myoglobin.…”