Motivation: Comparing two or more complex protein mixtures using liquid chromatography mass spectrometry (LC-MS) requires multiple analysis steps to locate and quantitate natural peptides within a single experiment and to align and normalize findings across multiple experiments. Results: We describe msInspect, an open-source application comprising algorithms and visualization tools for the analysis of multiple LC-MS experimental measurements. The platform integrates novel algorithms for detecting signatures of natural peptides within a single LC-MS measurement and combines multiple experimental measurements into a peptide array, which may then be mined using analysis tools traditionally applied to genomic array analysis. The platform supports quantitation by both label-free and isotopic labeling approaches. The software implementation has been designed so that many key components may be easily replaced, making it useful as a workbench for integrating other novel algorithms developed by a growing research community. Availability: The msInspect software is distributed freely under an Apache 2.0 license. The software as well as a Zip file with all peptide feature files and scripts needed to generate the tables and figures in this article are available at Contact:  mmcintos@fhcrc.org Supplementary Information: Supplementary materials are available at (select ‘Published Experiments’ from the list of Projects and then ‘msInspect Paper’).

Pathogenicity in Francisella tularensis subspecies

.Sequencing of the non-pathogenic Francisella tularensis sub-species novicida U112, and comparison with two pathogenic sub-species, provides insights into the evolution of pathogenicity in these species.

An integrated proteome concentration/separation approach involving on-line combination of capillary isoelectric focusing (CIEF) with capillary reversed-phase liquid chromatography (CRPLC) is developed for providing significant analyte concentration and extremely high resolving power toward protein and peptide mixtures. Upon completion of analyte focusing, the self-sharpening effect greatly restricts analyte diffusion and contributes to analyte stacking in narrowly focused bands with a concentration factor of approximately 240. In addition to analyte focusing, CIEF as the first separation dimension resolves proteins/peptides on the basis of their differences in pI and offers greater resolving power than that achieved in strong cation exchange chromatography. The grouping of two highly resolving and completely orthogonal separation techniques of CIEF and CRPLC, together with analyte focusing and concentration, significantly enhances the dynamic range and sensitivity of conventional mass spectrometry toward the identification of low-abundance proteins. The CIEF-based multidimensional separation/concentration platform enables the identification of a greater number of yeast soluble proteins than methods presented in the literature, yet requires a protein loading of only 9.6 microg. This protein loading is 2-3 orders of magnitude lower than those employed by the reported non-gel-based proteome techniques. The distribution of a codon adaptation index value for identified yeast proteins approximates to that predicted for the entire yeast proteome and supports the capability of CIEF-based proteome separation technology for achieving comprehensive proteome analysis. By reducing the inner diameter of chromatography columns from 180 microm to 100 microm, the required protein loading is further decreased from 9.6 microg to 960 ng, illustrating the potential usage of this proteome technology for the analysis of protein profiles within small cell populations or limited tissue samples.

A miniaturized trypsin membrane reactor housed inside a commonly used capillary fitting is developed and demonstrated for enabling rapid and sensitive protein identification by on-line proteolytic digestion and analysis of protein digests using nano-ESI-MS and MALDI-MS. The design and assembly of the capillary fitting-based trypsin membrane reactor are straightforward and highly robust, without the need for expensive fabrication technology and procedures. The resultant protein digests can also be further concentrated and resolved using capillary reversed-phase liquid chromatography or transient capillary isotachophoresis/zone electrophoresis prior to the mass spectrometric analysis in an integrated platform. By comparing these results with the results obtained from our previous studies using plastic microfluidics (Gao et al., Anal. Chem. 2001, 73, 2648-2655), significant reduction in dead volume and sample consumption can be achieved using this newly developed tryptic digestion station. This nanoscale reaction system enables rapid proteolytic digestion in seconds instead of hours for a protein concentration of less than 10(-8) M, consumes very little sample (< or = 5 fmol), and offers capillary interfaces with various separation and mass spectrometry techniques. The ultrafast enzymatic turnover for attaining complete peptide coverage in protein identification is contributed by the highly porous structure of the membrane media, providing excessive trypsin loading while eliminating the constraints of diffusion-limited reaction kinetics.