Biomedical subjects

William E Haskins

Publications and source records attributed to William E Haskins.

7 recordsLinked to original sources

PubMed2005

Unambiguous assignment of intramolecular chemical cross-links in modified mammalian membrane proteins by Fourier transform-tandem mass spectrometry.

Fourier transform tandem mass spectrometry (FT-MS/MS) can be used to unambiguously assign intramolecular chemical cross-links to specific amino acid residues even when two or more possible cross-linking sites are adjacent in the cross-linked protein. Bovine rhodopsin (Rho) in its dark-adapted state was intramolecularly cross-linked with lysine-cysteine (K-C) or lysine-lysine (K-K) cross-linkers to obtain interatomic distance information. Large, multiply charged, cross-linked peptide ions containing adjacent lysines, corresponding to Rho(50-86) (K(66) or K(67)) cross-linked to Rho(310-317) (C(316)) or Rho(318-348) (K(325) or K(339)), were fragmented by collision-induced dissociation (CID), infrared multiphoton dissociation (IRMPD), and electron capture dissociation (ECD). Complementary sequence-specific information was obtained by combining cross-link assignments; however, only ECD revealed full palmitoylation of adjacent cysteines (C(322) and C(323)) and cross-linking of K(67) (and not K(66)) to C(316), K(325), and K(339). ECD spectra contained crucial c- and z-ions resulting from cleavage of the bond between K(66) and K(67). To our knowledge, this work also presents the first demonstration that ECD can be used to characterize S-linked fatty acid acylation on cysteines. The comprehensive fragmentation of large peptides by CID, IRMPD, and particularly ECD, in conjunction with the high resolution and mass accuracy of FT-MS/MS, is shown to be a valuable means of characterizing mammalian membrane proteins with both chemical and posttranslational modifications.

Amino Acid Sequence↗

PubMed2005

A multidimensional differential proteomic platform using dual-phase ion-exchange chromatography-polyacrylamide gel electrophoresis/reversed-phase liquid chromatography tandem mass spectrometry.

Differential proteomic analysis has arisen as a large-scale means to discern proteome-wide changes upon treatment, injury, or disease. Tandem protein separation methods are required for large-scale differential proteomic analysis. Here, a novel multidimensional platform for resolving and differentially analyzing complex biological samples is presented. The platform, collectively termed CAX-PAGE/RPLC-MSMS, combines biphasic ion-exchange chromatography with polyacrylamide gel electrophoresis for protein separation, quantification, and differential band targeting, followed by capillary reversed-phase liquid chromatography and data-dependent tandem mass spectrometry for quantitative and qualitative peptide analysis. CAX-PAGE provides high protein resolving power with a theoretical peak capacity of 3570, extendable to 7600, a wide protein mass range verified from 16 to 273 kDa, and reproducible differential sample comparison without the added expense of fluorescent dyes and imaging equipment. Demonstrated using a neuroproteomic model, CAX-PAGE revealed an increased number of differential proteins, 137, compared with 82 found by 2D difference gel electrophoresis. When combined with RPLC-MSMS for protein identification, an additional quantification step is performed for internal validation, confirming a 2-fold or greater change in 89% of identified differential targets.

Animals↗

PubMed2005

Rapid discovery of putative protein biomarkers of traumatic brain injury by SDS-PAGE-capillary liquid chromatography-tandem mass spectrometry.

We report the rapid discovery of putative protein biomarkers of traumatic brain injury (TBI) by SDS-PAGE-capillary liquid chromatography-tandem mass spectrometry (SDS-PAGE-Capillary LC-MS(2)). Ipsilateral hippocampus (IH) samples were collected from naive rats and rats subjected to controlled cortical impact (a rodent model of TBI). Protein database searching with 15,558 uninterpreted MS(2) spectra, collected in 3 days via data-dependent capillary LC-MS(2) of pooled cyanine dye-labeled samples separated by SDS-PAGE, identified more than 306 unique proteins. Differential proteomic analysis revealed differences in protein sequence coverage for 170 mammalian proteins (57 in naive only, 74 in injured only, and 39 of 64 in both), suggesting these are putative biomarkers of TBI. Confidence in our results was obtained by the presence of several known biomarkers of TBI (including alphaII-spectrin, brain creatine kinase, and neuron-specific enolase) in our data set. These results show that SDS-PAGE prior to in vitro proteolysis and capillary LC-MS(2) is a promising strategy for the rapid discovery of putative protein biomarkers associated with a specific physiological state (i.e., TBI) without a priori knowledge of the molecules involved.

Amino Acid Sequence↗

PubMed2004

Discovery and neurochemical screening of peptides in brain extracellular fluid by chemical analysis of in vivo microdialysis samples.

Endogenous peptides from brain extracellular fluid of live rats were analyzed using capillary liquid chromatography (LC)-tandem mass spectrometry (MS2). A 4-mm-long microdialysis probe perfused at 0.6 microL/min implanted into the striatum of anesthetized male rats was used to collect 3.6 microL dialysate fractions that were injected on-line into the capillary LC-MS2 system for analysis. A total of 3349 MS2 spectra were collected from 13 different animals under basal conditions and during localized depolarization evoked by infusion of a high-K+ solution through the microdialysis probe. Subtractive analysis revealed a total of 859 MS2 spectra that were observed only during depolarization. From these spectra, 29 peptide sequences (25 were peptides not previously observed) from 6 different protein precursors were identified using database searching software. Proteins identified include precursors to neuropeptides, synaptic proteins, blood proteins, and transporters. The identified peptides represent candidates for neurotransmitters, neuromodulators, and markers of synaptic activity or brain tissue damage. A screen for neuroactivity of novel proenkephalin fragments that were found was performed by infusing the peptides into the brain while monitoring amino acid neurotransmitters by microdialysis sampling combined with capillary electrophoresis. Three of the six tested peptides evoked significant increases in various neuroactive amino acids. These results demonstrate that this combination of methods can identify novel neurotransmitter candidates and screen for potential neuroactivity.

Amino Acid Sequence↗

PubMed2004

Species differentiation of a diverse suite of Bacillus spores by mass spectrometry-based protein profiling.

In this study, we demonstrate the versatility of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOFMS) protein profiling for the species differentiation of a diverse suite of Bacillus spores. MALDI-TOFMS protein profiles of 11 different strains of Bacillus spores, encompassing nine different species, were evaluated. Bacillus species selected for MALDI-TOFMS analysis represented the spore-forming bacterial diversity of typical class 100K clean room spacecraft assembly facilities. A one-step sample treatment and MALDI-TOFMS preparation were used to minimize the sample preparation time. A library of MALDI-TOFMS spectra was created from these nine Bacillus species, the most diverse protein profiling study of the genus reported to date. Linear correlation analysis was used to successfully differentiate the MALDI-TOFMS protein profiles from all strains evaluated in this study. The MALDI-TOFMS protein profiles were compared with 16S rDNA sequences for their bacterial systematics and molecular phylogenetic affiliations. The MALDI-TOFMS profiles were found to be complementary to the 16S rDNA analysis. Proteomic studies of Bacillus subtilis 168 were pursued to identify proteins represented by the biomarker peaks in the MALDI-TOFMS spectrum. Four small, acid-soluble proteins (A, B, C, and D), one DNA binding protein, hypothetical protein ymf J, and four proteins associated with the spore coat and spore coat formation (coat JB, coat F, coat T, and spoIVA) were identified. The ability to visualize higher-molecular-mass coat proteins (10 to 25 kDa) as well as smaller proteins (<10 kDa) with MALDI-TOFMS profiling is critical for the complete and effective species differentiation of the Bacillus genus.

Bacillus↗

PubMed2002

In vivo neurochemical monitoring by microdialysis and capillary separations.

Microdialysis is valuable for studying the neurochemical changes underlying behavior. Recent advances include the application of the high-sensitivity methods of capillary electrophoresis and capillary liquid chromatography with mass spectrometry to dialysate analysis. These methods have improved temporal resolution, spatial resolution, multi-analyte capability and potential for compound discovery.

Animals↗

PubMedDate not supplied

Neuroproteomics in neurotrauma.

Neurotrauma in the form of traumatic brain injury (TBI) afflicts more Americans annually than Alzheimer's and Parkinson's disease combined, yet few researchers have used neuroproteomics to investigate the underlying complex molecular events that exacerbate TBI. Discussed in this review is the methodology needed to explore the neurotrauma proteome-from the types of samples used to the mass spectrometry identification and quantification techniques available. This neuroproteomics survey presents a framework for large-scale protein research in neurotrauma, as applied for immediate TBI biomarker discovery and the far-reaching systems biology understanding of how the brain responds to trauma. Ultimately, knowledge attained through neuroproteomics could lead to clinical diagnostics and therapeutics to lessen the burden of neurotrauma on society.

Animals↗