Studies of Posttranslational Modifications in Spiny Dogfish Myelin Basic Protein

The objective of this investigation was to determine whether nonmammalian myelin basic protein contained charge isomers resulting from extensive posttranslational modifications as seen in mammalian MBP. Four charge isomer components from dogfish MBP have been isolated. These forms arise by phosphorylation and deamidation modifications. Components C1, C2 and C3 have been characterized. We are currently characterizing component C8. Dogfish MBP is less cationic than mammalian MBP and has about 50% lower mobility on a basic pH gel electrophoresis relative to human and to bovine MBP. The mammalian component C1, which is unmodified, is modified in the dogfish by phosphorylation. The reduced electrophoretic mobility is largely attributable to the charge reduction resulting from phosphorylation in serine 72, 83, and 120 or 121 in C1, and C3. In component C2, two or three phosphate groups were distributed among residues 134, 138 and 139. It was found that dogfish amino acid residue 30 was a lysine residue and not a glutamate residue as reported in the literature.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45421/1/11064_2004_Article_344513.pd

Survival and Passage of Yearling Chinook Salmon and Steelhead at The Dalles Dam, Spring 2011 - FINAL REPORT

The study reported herein was conducted by the Pacific Northwest National Laboratory (PNNL) and the University of Washington (UW) for the U.S. Army Corps of Engineers, Portland District (USACE). The PNNL and UW project managers were Drs. Thomas J. Carlson and John R. Skalski, respectively. The USACE technical lead was Mr. Brad Eppard. The study was designed to estimate dam passage survival and other performance measures at The Dalles Dam as stipulated by the 2008 Federal Columbia River Power System Biological Opinion (BiOp) and the 2008 Columbia Basin Fish Accords. The study is being documented in two types of reports: compliance and technical. A compliance report is delivered within 6 months of the completion of the field season and focuses on results of the performance metrics outlined in the 2008 BiOp and Fish Accords. A technical report is produced within the 18 months after field work, providing comprehensive documentation of a given study and results on route-specific survival estimates and fish passage distributions, which are not included in compliance reports. This technical report concerns the 2011 acoustic telemetry study at The Dalles Dam

Application of on-line capillary electrophoresis /tandem mass spectrometry to the study of myelin basic proteins.

On-line capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) of posttranslational modifications (PTMs) of Myelin Basic Protein (MBP) charge isomer tryptic digests has been utilized to investigate spiny dogfish and human MBPs. CE has permitted rapid separations of mixtures with high resolution. The quadrupole ion trap storage/reflectron time-of-flight mass spectrometer has proven to be an efficient detection device for the CE eluent of tryptic peptides from spiny dogfish and human MBPs. The comparison of PTM of MBP from spiny dogfish and humans provides information for understanding the evolutionary process from nonmammalian vertebrate (spiny dogfish) to mammalian vertebrate (human). The current results demonstrate the presence of PTM in both spiny dogfish and human MBP and the connection between these two MBPs. The PTM sites on normal and Multiple Sclerosis forms of human MBP charge isomers have been investigated using the CE-MS/MS system. Multiple Sclerosis patients showed more extensive methylation than healthy persons on the arginine at residue 107 in charge isomers c4 and c5. Phosphorylation appeared much more frequently and intensively in normal than Multiple Sclerosis charge isomers. At the present time it is thought that the phosphorylation of MBP plays an important role in maintaining the tight wrappings of the myelin structure, and dephosphorylation facilitates the destabilization of myelin and permits attack by the immune system, which results in Multiple Sclerosis. Also, excellent correlation was observed for electrophoretic mobilities of tryptic peptides of human MBP with q/M0.56, where q is the calculated net charge and M is the molecular weight. Posttranslationally modified peptides also followed this correlation.Ph.D.Analytical chemistryPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/129767/2/3042098.pd

Characterization of Unknown Compounds from Stainless Steel Plates in Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

Peaks originating from unknown compounds on stainless steel plates used in matrix-assisted laser desorption/ionization (MALDI) mass spectrometers are observed around m/z 304.3, 332.3, 360.4, and 388.4 regardless of the matrix and/or solvent, and are even observed with bare plates. These peaks were characterized using three different types of MALDI-MS instrumentation: MALDI-TOF MS, MALDI-TOF/TOF MS, and MALDI-FTMS. The fragmentation data from MALDI-TOF/TOF MS and accurate mass determination by MALDI-FTMS enabled identification of the chemical formulae and structures. The unknown compounds are, in fact, likely benzylalkylmethylammonium salts, as confirmed by closely matching fragmentation patterns with a commercially available benzalkonium chloride

Microwave-assisted Weak Acid Hydrolysis of Proteins

Myoglobin was hydrolyzed by microwave-assisted weak acid hydrolysis with 2% formic acid at 37 oC, 50 oC, and100 oC for 1 h. The most effective hydrolysis was observed at 100 oC. Hydrolysis products were investigated using matrixassistedlaser desorption/ionization time-of-flight mass spectrometry. Most cleavages predominantly occurred at the C-termini ofaspartyl residues. For comparison, weak acid hydrolysis was also performed in boiling water for 20, 40, 60, and 120 min. A 60-min weak acid hydrolysis in boiling water yielded similar results as a 60-min microwave-assisted weak acid hydrolysis at100 oC. These results strongly suggest that microwave irradiation has no notable enhancement effect on acid hydrolysis of proteinsand that temperature is the major factor that determines the effectiveness of weak acid hydrolysis

Extended Range Proteomic Analysis (ERPA): A New and Sensitive LC-MS Platform for High Sequence Coverage of Complex Proteins with Extensive Post-translational Modifications - Comprehensive Analysis of Beta-Casein and Epidermal Growth Factor Receptor (EGFR)

16 page(s

Commercial Silicon-on-Insulator (SOI) Wafers as a Versatile Substrate for Laser Desorption/Ionization Mass Spectrometry

We report here that a commercial silicon-on-insulator (SOI) wafer offers an opportunity for laser desorption/ionization (LDI) of peptide molecules, which occurs directly from its flat surface without requiring special surface preparation. The LDI-on-SOI exhibits intact ionization of peptides with a good detection limit of lower than 20 fmol, of which the mass range is demonstrated up to insulin with citric acid additives. The LDI process most likely arises from laser-induced surface heating promoted by two-dimensional thermal confinement in the thin Si surface layer of the SOI wafer. As a consequence of the thermal process, the LDI-on-SOI method is also capable of creating post-source decay (PSD) of the resulting peptide LDI ions, which is suitable for peptide sequencing using conventional TOF/TOF mass spectrometry. © 2012 American Society for Mass Spectrometry.

Matrix-assisted laser desorption/ionization-Fourier-transform ion cyclotron resonance-mass spectrometry analysis of exosomal lipids from human serum

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/175182/1/rcm9427.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/175182/2/rcm9427_am.pd