Femia Hopwood

Automatic Poisson peak harvesting for high throughput protein identification

High throughput identification of proteins by peptide mass fingerprinting requires an efficient means of picking peaks from mass spectra. Here, we report the development of a peak harvester to automatically pick monoisotopic peaks from spectra generated on matrix‐assisted laser desorption/ionisation time of flight (MALDI‐TOF) mass spectrometers. The peak harvester uses advanced mathematical morphology and watershed algorithms to first process spectra to stick representations. Subsequently, Poisson modelling is applied to determine which peak in an isotopically resolved group represents the monoisotopic mass of a peptide. We illustrate the features of the peak harvester with mass spectra of standard peptides, digests of gel‐separated bovine serum albumin, and with Escherictia coli proteins prepared by two‐dimensional polyacrylamide gel electrophoresis. In all cases, the peak harvester proved effective in its ability to pick similar monoisotopic peaks as an experienced human operator, and also proved effective in the identification of monoisotopic masses in cases where isotopic distributions of peptides were overlapping. The peak harvester can be operated in an interactive mode, or can be completely automated and linked through to peptide mass fingerprinting protein identification tools to achieve high throughput automated protein identification.

High Throughput Peptide Mass Fingerprinting and Protein Macroarray Analysis Using Chemical Printing Strategies

We describe a chemical printer that uses piezoelectric pulsing for rapid, accurate, and non-contact microdispensing of fluid for proteomic analysis of immobilized protein macroarrays. We demonstrate protein digestion and peptide mass fingerprinting analysis of human plasma and platelet proteins direct from a membrane surface subsequent to defined microdispensing of trypsin and matrix solutions, hence bypassing multiple liquid-handling steps. Detection of low abundance, alkaline proteins from whole human platelet extracts has been highlighted. Membrane immobilization of protein permits archiving of samples pre-/post-analysis and provides a means for subanalysis using multiple chemistries. This study highlights the ability to increase sequence coverage for protein identification using multiple enzymes and to characterize N-glycosylation modifications using a combination of PNGase F and trypsin. We also demonstrate microdispensing of multiple serum samples in a quantitative microenzyme-linked immunosorbent assay format to rapidly screen protein macroarrays for pathogen-derived antigens. We anticipate the chemical printer will be a major component of proteomic platforms for high throughput protein identification and characterization with widespread applications in biomedical and diagnostic discovery.

Automated Peak Harvesting of MALDI-MS spectra for high throughput proteomics

High throughput proteomics is realized not only by the use of automated hardware but also by the application of efficient, automated software routines to complex data. In this paper, we present the recent developments of our software tool Peak Harvester for the automatic harvesting of monoisotopic peaks from MALDI-TOF mass spectra of peptides. Peak Harvester uses advanced mathematical morphology to convert mass spectra into stick representations. Poisson modeling of theoretical isotopic distributions is then applied to derive the monoisotopic peptide mass from an isotopically resolved group of peaks. The accuracy of Peak Harvester is demonstrated via the analysis of peptide spectra from low concentrations of bovine serum albumin blotted onto PVDF membranes and of tryptic digested platelet proteins derived from human blood following two- dimensional gel electrophoresis. The results demonstrate the power of this software as it can accurately assign monoisotopic masses, including those from overlapping isotopic distributions, and picks masses as accurately as an experienced human operator. We have further developed Peak Harvester to include peak harvesting from MALDI-TOF Post Source Decay (PSD) experiments. Here we demonstrate the versatility of the software by both the analysis of PSD data from 2DE and the analysis of peptide mass spectra collected directly from tryptic digests on a PVDF membrane.