Rapid identification of proteins by peptide-mass fingerprinting

The amino acid sequence of the glucose transport protein from human HepG2 hepatoma cells was deduced from analysis of a complementary DNA clone. Structural analysis of the purified human erythrocyte glucose transporter by fast atom bombardment mapping and gas phase Edman degradation confirmed the identity of the clone and demonstrated that the HepG2 and erythrocyte transporters are highly homologous and may be identical. The protein lacks a cleavable amino-terminal signal sequence. Analysis of the primary structure suggests the presence of 12 membrane-spanning domains. Several of these may form amphipathic alpha helices and contain abundant hydroxyl and amide side chains that could participate in glucose binding or line a transmembrane pore through which the sugar moves. The amino terminus, carboxyl terminus, and a highly hydrophilic domain in the center of the protein are all predicted to lie on the cytoplasmic face. Messenger RNA species homologous to HepG2 glucose transporter messenger RNA were detected in K562 leukemic cells, HT29 colon adenocarcinoma cells, and human kidney tissue.

http://science.sciencemag.org/content/sci/229/4717/941.full.pdf

Sequence and structure of a human glucose transporter

4. Automated Interpretation of CID Spectra 282 5. Accurate Mass Tags 282 C. Protein Identification in Complex Mixtures 282 D. Analysis of Protein Expression 284 III. Proteomes and Post-Translational Modifications 285 A. Proteomes 285 1. The Analytical Challenge 285 2. Analysis of Protein−Protein Complexes 286 B. Post-Translational Modifications 286 1. Background 286 2. Detection and Purification of Phosphoproteins 288

Mass Spectrometry in Proteomics

NMR spectroscopy in the structural elucidation of oligosaccharides and glycosides.

Desorption mass spectrometry has undergone significant improvements since the original experiments were performed more than 90 years ago1. The most dramatic change occurred in the early1980s with the introduction of an organic matrix2,3,4 to transfer energy to the analyte. This reduces ion fragmentation but also introduces background ions from the matrix. Here we describe a matrix-free strategy forbiomolecular mass spectrometry based on pulsed-laser desorption–ionization from a porous silicon5 surface. Our method uses porous silicon to trap analytes deposited on the surface, and laser irradiation to vaporize and ionize them. We show that the method works at femtomole and attomole levels of analyte, and induces little or no fragmentation, in contrast to what is typically observed with other such approaches6,7,8,9,10,11. The ability to perform these measurements without a matrix3,4,12,13 also makes itmore amenable to small-molecule analysis. Chemical14 and structural15 modification of the porous silicon has enabled optimization of the ionization characteristics of the surface. Our technique offers good sensitivity as well as compatibility with silicon-based microfluidics and microchip technologies.

Desorption–ionization mass spectrometry on porous silicon

A new method for obtaining high-quality mass spectra of molecules which previously were difficult or impossible to study by ionisation methods is described

Fast atom bombardment of solids (F.A.B.): a new ion source for mass spectrometry

A new ion source for molecular structure determination of thermolabile and involatile compounds by mass spectrometry is presented together with some preliminary results from peptides, glycoside antibiotics, organometallics and vitamin B12 its coenzyme.

Fast atom bombardment of solids as an ion source in mass spectrometry

Fast atom bombardment: A new mass spectrometric method for peptide sequence analysis

The positive- and negative-ion mass spectra of [methionine]enkephalin and [leucine]enkephalin have been obtained by using a fast-atom-bombardment source described previously by Barber, Bordoli, Sedgwick & Tyler [(1981) J. Chem. Soc. Chem. Commun., in the press]. This technique has allowed the spectra to be obtained without conversion of the enkephalins into volatile derivatives. The fast-atom-bombardment spectra show good pseudo-molecular-ion sensitivity and fragmentation that can be interpreted on the basis of the known molecular structure.

Fast-atom-bombardment mass spectra of enkephalins.

The new technique of fast atom bombardment mass spectrometry has been applied to bradykinin, lysyl-bradykinin and methionyl-lysyl-bradykinin. High sensitivity was obtained at [M + H]+ in the positive ion spectra with a comparable response at [M—H]− in the opposite polarity. All spectra contained an abundance of structurally significant fragment ions. Schemes for relating the observed fragments to the known amino acid sequences are reported for all three molecules. Mass spectrometric investigations on bradykinin were followed by quantitative bioassays which showed that atom bombardment for ten minutes had no measurable effect on the biological activity of microgram samples.

Andrew N. Tyler

Papers

Fast atom bombardment of solids (F.A.B.): a new ion source for mass spectrometry

Fast atom bombardment of solids as an ion source in mass spectrometry

Fast atom bombardment: A new mass spectrometric method for peptide sequence analysis

Fast-atom-bombardment mass spectra of enkephalins.

Fast atom bombardment mass spectrometry of bradykinin and related oligopeptides