Mass Spectrometry For Peptide Analysis Research Articles

Analysis of protein expression in Brucella abortus mutants with different growth rates by two-dimensional gel electrophoresis and LC-MS/MS peptide analysis.

Brucella abortus is a bacterium that causes brucellosis and is the causative agent of worldwide zoonoses. Pathogenesis of the B. abortus infection is complicated, and several researchers have attempted to elucidate the infection mechanism of B. abortus. While several proteins have been revealed as pathogenic factors by previous researchers, the underlying mechanism of B. abortus infection is unresolved. In this study, we identified proteins showing different expression levels in B. abortus mutants with different biological characteristics that were generated by random insertion of a transposon. Five mutants were selected based on biological characteristics, in particular, their growth features. Total proteins of mutant and wild-type B. abortus were purified and subjected to two-dimensional gel electrophoresis. Thirty protein spots of each mutant with expression increases or decreases were selected; those with a change of more than 2-fold were compared with the wild-type. Selected spots underwent liquid chromatography tandem mass spectrometry for peptide analysis. DnaK and ClpB, involved in protein aggregation, increased. SecA and GAPDH, associated with energy metabolism, decreased in some mutants with a growth rate slower than that of the wild-type. Mutants with slower growth showed a decrease in energy metabolism-related proteins, while mutants with faster growth showed an increase in pathogenicity-related proteins.

Miniaturized Ultra High Field Asymmetric Waveform Ion Mobility Spectrometry Combined with Mass Spectrometry for Peptide Analysis

Miniaturized ultra high field asymmetric waveform ion mobility spectrometry (ultra-FAIMS) combined with mass spectrometry (MS) has been applied to the analysis of standard and tryptic peptides, derived from α-1-acid glycoprotein, using electrospray and nanoelectrospray ion sources. Singly and multiply charged peptide ions were separated in the gas phase using ultra-FAIMS and detected by ion trap and time-of-flight MS. The small compensation voltage (CV) window for the transmission of singly charged ions demonstrates the ability of ultra-FAIMS-MS to generate pseudo-peptide mass fingerprints that may be used to simplify spectra and identify proteins by database searching. Multiply charged ions required a higher CV for transmission, and ions with different amino acid sequences may be separated on the basis of their differential ion mobility. A partial separation of conformers was also observed for the doubly charged ion of bradykinin. Selection on the basis of charge state and differential mobility prior to tandem mass spectrometry facilitates peptide and protein identification by allowing precursor ions to be identified with greater selectivity, thus reducing spectral complexity and enhancing MS detection.

Open-Tubular Capillary Electrochromatography Coupled with Electrospray Ionization Mass Spectrometry for Peptide Analysis

In this study, the open-tubular electrochromatographic (OT-CEC) migration behavior of various peptides has been characterized using etched and chemically (n-octadecyl- and cholesterol-) modified capillaries, interfaced to an electrospray ionization mass spectrometer through a sheath liquid configuration. The stationary phases were fabricated by etching the inner wall of the fused-silica capillary and then chemically modifying the new surface through a silanization/hydrosilation reaction. Unlike some other OT-CEC stationary-phase preparation methods, leaching of the immobilized stationary phase and subsequent contamination of the electrospray ion source was largely avoided with this novel surface modification technology. The influence of the immobilized organic phases and those of the buffer electrolytes (pH, the type and content of organic solvent) on the retention and separation of the selected peptides was investigated. Significant peptide retention was found even at very low pH with both types of stationary phases, under conditions whereby the electrophoretic migration dominated the separation process. Due to the effective coverage of the etched surface by a silanization/hydrosilation reaction, adverse adsorption of charged analytes onto the capillary wall was minimized. As a result, very efficient and highly reproducible peptide separations were achieved over a broad pH range. Moreover, peptide-specific multizoning effects were observed. The origin of this novel phenomenon was explored. Compared to capillary electrophoresis electrospray ionization mass spectrometry system, much higher detection sensitivity could be obtained, since a larger amount of sample could be injected and stacked at the head of the open-tubular capillary column without deteriorating the separation performance. On the basis of these observations, these procedures have been adapted to allow the analysis of tryptic peptides generated from proteins.

Proteomic Analysis with Integrated Multiple Dimensional Liquid Chromatography/Mass Spectrometry Based on Elution of Ion Exchange Column Using pH Steps

A novel integrated multidimensional liquid chromatography (IMDL) method is demonstrated for the separation of peptide mixtures by two-dimensional HPLC coupled with ion trap mass spectrometry. The method uses an integrated column, containing both strong cation exchange and reversed-phase sections for two-dimensional liquid chromatography. The peptide mixture was fractionated by a pH step using a series of pH buffers, followed by reversed-phase chromatography. Since no salt was used during separation, the integrated multidimensional liquid chromatography can be directly connected to mass spectrometry for peptide analysis. The pH buffers were injected from an autosampler, and the entire process can be carried out on a one-dimensional liquid chromatography system. In a single analysis, the IMDL system, coupled with linear ion trap mass spectrometry, identified more than 2000 proteins in mouse liver. The peptides were eluted according to their pI distribution. The resolution of the pH fractionation is approximately 0.5 pH unit. The method has low overlapping across pH fractions, good resolution of peptide mixture, and good correlation of peptide pIs with pH steps. This method provides a technique for large-scale protein identification using existing one-dimensional HPLC systems.