Biomedical subjects

Michael S Westphall

Publications and source records attributed to Michael S Westphall.

4 recordsLinked to original sources

PubMed2006

Identification of mammalian cell lines using MALDI-TOF and LC-ESI-MS/MS mass spectrometry.

Direct mass spectrometric analysis of complex biological samples is becoming an increasingly useful technique in the field of proteomics. Matrix-assisted laser desorption/ionization mass spectroscopy (MALDI-MS) is a rapid and sensitive analytical tool well suited for obtaining molecular weights of peptides and proteins from complex samples. Here, a fast and simple approach to cellular protein profiling is described in which mammalian cells are lysed directly in the MALDI matrix 2,5-dihydroxybenzoic acid (DHB) and mass analyzed using MALDI-time of flight (TOF). Using the unique MALDI mass spectral "fingerprint" generated in these analyses, it is possible to differentiate among several different mammalian cell lines. A number of techniques, including MALDI-post source decay (PSD), MALDI tandem time-of-flight (TOF-TOF), MALDI-Fourier transform ion cyclotron resonance (FTICR), and nanoflow liquid chromatography followed by electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) were employed to attempt to identify the proteins represented in the MALDI spectra. Performing a tryptic digestion of the supernatant of the cells lysed in DHB with subsequent LC-ESI-MS/MS analysis was by far the most successful method to identify proteins.

Amino Acid Sequence↗

PubMed2005

Controlling gas-phase reactions for efficient charge reduction electrospray mass spectrometry of intact proteins.

Charge reduction electrospray mass spectrometry (CREMS) reduces the charge states of electrospray-generated ions, which concentrates the ions from a protein into fewer peaks spread over a larger m/z range, thereby increasing peak separation and decreasing spectral congestion. An optimized design for a CREMS source is described that provides an order-of-magnitude increase in sensitivity compared to previous designs and provides control over the extent of charge reduction. Either a corona discharge or an alpha-particle source was employed to generate anions that abstract protons from electrosprayed protein cations. These desired ion/ion proton transfer reactions predominated, but some oxidation and ion-attachment reactions also occurred, leading to new peaks or mass-shifted broader peaks while decreasing signal intensity. The species producing these deleterious side-reactions were identified, and conditions were found that prevented their formation. Spectrometer m/z biases were examined because of their effect upon the signal intensity of higher m/z charge-reduced protein ions. The utility of this atmospheric pressure CREMS was demonstrated using a cell lysate fraction from E. coli. The spectral simplification afforded by CREMS reveals more proteins than are observed without charge reduction.

Electrochemistry↗

PubMed2003

Mass spectrometric analysis of DNA mixtures: instrumental effects responsible for decreased sensitivity with increasing mass.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry has demonstrated great potential to replace gel electrophoresis for DNA sequence analysis. A current limitation in this method is, however, the decreased sensitivity with increasing mass of DNA molecules. In the present study, instrumental effects on the mass analysis of DNA molecules were investigated quantitatively using an equimolar DNA mixture. It is shown that detection efficiency, detector saturation, and ion beam divergence account for the entirety of the observed falloff in signal intensity with increasing mass. Although the present study focused upon the analysis of DNA mixtures, the instrumental effects observed apply equally to other macromolecular mixtures (e.g., proteins, polymers).

Algorithms↗

PubMed2002

Single-pulse nanoelectrospray ionization.

A new electrospray ionization (ESI) source that provides a means of generating single packets of ions for mass spectrometric analysis is presented. Sample solution held at a high potential is ejected from a glass capillary with a small dispensing aperture (20-microm i.d.) by constriction of a cylindrical piezoelectric element. Unlike conventional ESI sources that are continuous, this source dispenses fixed volumes of solution as small as 10 pL and provides detection sensitivity in the attomole range when coupled to an orthogonal time-of-flight mass spectrometer. In addition to picoliter-level control over the dispensed volume, the source permits control of the frequency with which ionization pulses are generated as well as the ability to start and stop the pulses without altering the applied solution potential. The source was characterized by analysis of both protein and DNA samples from a variety of different solution compositions. This source design should be compatible with virtually any ESI mass analyzer.

DNA↗