Benjamin J. Cargile

Ion trap collisional activation of disulfide linkage intact and reduced multiply protonated polypeptides

The presence of disulfide linkages in multiply charged polypeptide ions tends to inhibit the formation of structurally informative product ions under conventional quadrupole ion trap collisional activation conditions. In particular, fragmentation that requires two cleavages (i.e., cleavage of a disulfide linkage and a peptide linkage) is strongly suppressed. Reduction of the disulfide linkage(s) by use of dithiothreitol yields parent ions upon electrospray without this complication. Far richer structural information is revealed by ion trap collisional activation of the disulfide-reduced species than from the native species. These observations are illustrated with doubly protonated native and reduced somatosin, the [M + 5H](5+) ion of native bovine insulin and the [M + 4H](4+) and [M + 3H](3+) ions of the B-chain of bovine insulin produced by reduction of the disulfide linkages in insulin, and the [M + 11H](11+) ion of native chicken lysozyme and the [M + 11H](11+) and [M + 14H](14+) ions of reduced lysozyme. In each case, the product ions produced by ion trap collisional activation were subjected to ion/ion proton transfer reactions to facilitate interpretation of the product ion spectra. These studies clearly suggest that the identification of polypeptides with one or more disulfide linkages via application of ion trap collisional activation to the multiply charged parent ions formed directly by electrospray could be problematic. Means for cleaving the disulfide linkage, such as reduction by dithiothreitol prior to electrospray, are therefore desirable in these cases.

Automated Proteomics of E. coli via Top-Down Electron-Transfer Dissociation Mass Spectrometry

Electron-transfer dissociation (ETD) has recently been introduced as a fragmentation method for peptide and protein analysis. Unlike collisionally induced dissociation (CID), fragmentation by ETD occurs randomly along the peptide backbone. With the use of the sequences determined from the protein termini and the parent protein mass, intact proteins can be unambiguously identified. Because of the fast kinetics of these reactions, top-down proteomics can be performed using ETD in a linear ion trap mass spectrometer on a chromatographic time scale. Here we demonstrate the utility of ETD in high-throughput top-down proteomics using soluble extracts of E. coli. Development of a multidimensional fractionation platform, as well as a custom algorithm and scoring scheme specifically designed for this type of data, is described. The analysis resulted in the robust identification of 322 different protein forms representing 174 proteins, comprising one of the most comprehensive data sets assembled on intact proteins to date.

Identification of Quantitative Trait Loci Underlying Proteome Variation in Human Lymphoblastoid Cells

Population-based variability in protein expression patterns, especially in humans, is often observed but poorly understood. Moreover, very little is known about how interindividual genetic variation contributes to protein expression patterns. To begin to address this, we describe elements of technical and biological variations contributing to expression of 544 proteins in a population of 24 individual human lymphoblastoid cell lines that have been extensively genotyped as part of the International HapMap Project. We determined that expression levels of 10% of the proteins were tightly correlated to cell doubling rates. Using the publicly available genotypes for these lymphoblastoid cell lines, we applied a genetic association approach to identify quantitative trait loci associated with protein expression variation. Results identified 24 protein forms corresponding to 15 proteins for which genetic elements were responsible for >50% of the expression variation. The genetic variation associated with protein expression levels were located in cis with the gene coding for the transcript of the protein for 19 of these protein forms. Four of the genetic elements identified were coding non-synonymous single nucleotide polymorphisms that resulted in migration pattern changes in the two-dimensional gel. This is the first description of large scale proteomics analysis demonstrating the direct relationship between genome and proteome variations in human cells.