Integration of mass spectrometry in analytical biotechnology.
Mass spectrometry (MS) has become an indispensable tool for peptide and protein structure analysis because of three unique capabilities that enable it to be used to solve structural problems not easily handled by conventional techniques. First, MS is able to provide accurate molecular weight information on low-picomole amounts of peptides and proteins independent of covalent modifications that may be present. Second, this information is obtainable for peptides present in complex mixtures such as those that result from a proteolytic digest of a protein. Third, by using tandem MS, partial to complete sequence information may be obtained for peptides containing up to 25 amino acid residues, even if the peptides are present in mixtures. Sensitivity and speed of the MS-based approaches now equal (and in some cases exceed) that of Edman-based sequence analysis. In this perspective we discuss how MS, tandem high-performance MS, and on-line liquid chromatography/MS using fast atom bombardment or electrospray ionization have been integrated with more conventional techniques in order to increase the accuracy and speed of peptide and protein structure characterization. The expanding role of matrix-assisted laser desorption MS in protein analysis is also described. The unique niche that MS occupies for locating and structurally characterizing posttranslational modifications of proteins is emphasized. Examples chosen from the authors' laboratory illustrate how MS is used to sequence blocked proteins, define N- and C-terminal sequence heterogeneity, locate and correct errors in DNA- and cDNA-deduced protein sequences, identify sites of deamidation, isoaspartyl formation, phosphorylation, oxidation, disulfide bond formation, and glycosylation, and define the structural class of carbohydrate at specific attachment sites in glycoproteins.
Carr
S A
SA
Department of Physical and Structural Chemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406.
Hemling
M E
ME
Bean
M F
MF
Roberts
G D
GD
D001709:Biotechnology / Q000295:instrumentation*; D013058:Mass Spectrometry; D010455:Peptides / Q000032:analysis; D011506:Proteins / Q000032:analysis
D010455:Peptides; D011506:Proteins