Online two-dimensional porous graphitic carbon/reversed phase liquid chromatography platform applied to shotgun proteomics and glycoproteomics.

A novel fully automatable two-dimensional liquid chromatography (2DLC) platform has been integrated into a modified commercial off-the-shelf LC instrument, incorporating porous graphitic carbon (PGC) separation and conventional low-pH reversed-phase (RP) separation for both proteomics and N-glycomics analyses; the dual-trap column configuration of this platform offers desirable high-throughput analyses with almost no idle time, in addition to a miniaturized setup and simplified operation. The total run time per analysis was only 19 h when using eight PGC fractions for unattended large-scale qualitative and quantitative proteomic analyses; the identification of 2678 nonredundant proteins and 11,984 unique peptides provided one of the most comprehensive proteome data sets for primary cerebellar granule neurons (CGNs). The effect of pH on the PGC column was investigated for the first time to improve the hydrophobic peptide coverage; the performance of the optimized system was first benchmarked using tryptic digests of Saccharomyces cerevisiae cell lysates and then evaluated through duplicate analyses of Macaca fascicularis cerebral cortex lysates using isobaric tags for relative and absolute quantitation (iTRAQ) technology. An additional plug-and-play PGC module functioned in a complementary manner to recover unretained hydrophilic solutes from the low-pH RP column; synchronization of the fractionations between the PGC-RP system and the PGC module facilitated simultaneous analyses of hydrophobic and hydrophilic compounds from a single sample injection event. This methodology was applied to perform, for the first time, detailed glycomics analyses of Macaca fascicularis plasma, resulting in the identification of a total 130 N-glycosylated plasma proteins, 705 N-glycopeptides, and 254 N-glycosylation sites.

Fully automatable multidimensional reversed-phase liquid chromatography with online tandem mass spectrometry.

Liquid chromatography (LC) is essential for sample fractionation in shotgun proteomics applications. With suitable design, common LC separation chemistries, including reversed-phase (RP) and strong cation exchange (SCX) mode, can be combined in online multidimensional LC to greatly enhance the overall separation power and, thus, proteome coverage. This protocol describes the design and assembly of a flexible online multidimensional RP-SCX-RP LC system that is compatible with deep proteome profiling on common shotgun proteomics platforms.

Novel Cß-Cγ bond cleavages of tryptophan-containing peptide radical cations.

In this study, we observed unprecedented cleavages of the C(ß)-C(γ) bonds of tryptophan residue side chains in a series of hydrogen-deficient tryptophan-containing peptide radical cations (M(•+)) during low-energy collision-induced dissociation (CID). We used CID experiments and theoretical density functional theory (DFT) calculations to study the mechanism of this bond cleavage, which forms [M - 116](+) ions. The formation of an α-carbon radical intermediate at the tryptophan residue for the subsequent C(ß)-C(γ) bond cleavage is analogous to that occurring at leucine residues, producing the same product ions; this hypothesis was supported by the identical product ion spectra of [LGGGH - 43](+) and [WGGGH - 116](+), obtained from the CID of [LGGGH](•+) and [WGGGH](•+), respectively. Elimination of the neutral 116-Da radical requires inevitable dehydrogenation of the indole nitrogen atom, leaving the radical centered formally on the indole nitrogen atom ([Ind](•)-2), in agreement with the CID data for [WGGGH](•+) and [W(1-CH3)GGGH](•+); replacing the tryptophan residue with a 1-methyltryptophan residue results in a change of the base peak from that arising from a neutral radical loss (116 Da) to that arising from a molecule loss (131 Da), both originating from C(ß)-C(γ) bond cleavage. Hydrogen atom transfer or proton transfer to the γ-carbon atom of the tryptophan residue weakens the C(ß)-C(γ) bond and, therefore, decreases the dissociation energy barrier dramatically.