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Capillary liquid chromatography coupled with an ion trap storage/reflectron time-of-flight mass spectrometer for structural confirmation of three recombinant protein isoforms. 1996

M G Qian, and K Zheng, and Y Chen, and C L Chang, and S M Hanash, and D M Lubman
Department of Chemistry, University of Michigan, Ann Arbor 48109, USA.

Packed-capillary high-performance liquid chromatography (HPLC) was successfully coupled with an ion trap storage/reflectron time-of-flight mass spectrometer (LC/IT/reTOFMS) through an electrospray ionization interface for protein structural elucidation. Using the total-ion storage capabilities of the trap over a broad mass range and the high sensitivity from the packed capillary column with i.d. as small as 250 microns, high sensitivity peptide mapping in the low picomole range was demonstrated for the structural confirmation of three recombinant human nucleoside diphosphate kinase isoforms (NDPK, E.C. 2.7.4.6). A strategy combining chemical/enzymatic digestions as well as collisionally-induced dissociation (CID) in the electrospray source was successfully employed to infer the minor primary structural differences among the three recombinant proteins. This high sensitivity was achieved while also maintaining a resolution of nearly 1500 for mass identification using the capabilities of the IT/reTOF device. A point mutation of serine 120 to glycine was verified between the wild-type NDPK A and its mutant (delta m = 30 u) by both selected-ion monitoring and ion-source CID of the protein fragment containing the mutation site. For the structural confirmation of the sequence of NDPK A and B (88% homology), two sets of chemical/proteolytic digests were generated independently and followed by LC/MS analysis of the molecular weight of each protein-generated fragment. The complementary information from the two chromatographic analyses allowed for sequence verification of the two protein isoforms. The experiments clearly demonstrated that the high concentration sensitivity of the capillary high-performance liquid chromatographic separation together with the advantages of the IT/reTOF mass spectrometer could provide a low-cost, high-performance facility for protein analysis.

UI MeSH Term Description Entries
D007527 Isoenzymes Structurally related forms of an enzyme. Each isoenzyme has the same mechanism and classification, but differs in its chemical, physical, or immunological characteristics. Alloenzyme,Allozyme,Isoenzyme,Isozyme,Isozymes,Alloenzymes,Allozymes
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D009701 Nucleoside-Diphosphate Kinase An enzyme that is found in mitochondria and in the soluble cytoplasm of cells. It catalyzes reversible reactions of a nucleoside triphosphate, e.g., ATP, with a nucleoside diphosphate, e.g., UDP, to form ADP and UTP. Many nucleoside diphosphates can act as acceptor, while many ribo- and deoxyribonucleoside triphosphates can act as donor. EC 2.7.4.6. Deoxynucleoside Diphosphate Kinases,GDP Kinase,Nucleoside Diphosphokinases,Nucleoside-Diphosphate Kinases,Diphosphate Kinases, Deoxynucleoside,Diphosphokinases, Nucleoside,Kinase, GDP,Kinase, Nucleoside-Diphosphate,Kinases, Deoxynucleoside Diphosphate,Kinases, Nucleoside-Diphosphate,Nucleoside Diphosphate Kinase,Nucleoside Diphosphate Kinases
D010449 Peptide Mapping Analysis of PEPTIDES that are generated from the digestion or fragmentation of a protein or mixture of PROTEINS, by ELECTROPHORESIS; CHROMATOGRAPHY; or MASS SPECTROMETRY. The resulting peptide fingerprints are analyzed for a variety of purposes including the identification of the proteins in a sample, GENETIC POLYMORPHISMS, patterns of gene expression, and patterns diagnostic for diseases. Fingerprints, Peptide,Peptide Fingerprinting,Protein Fingerprinting,Fingerprints, Protein,Fingerprint, Peptide,Fingerprint, Protein,Fingerprinting, Peptide,Fingerprinting, Protein,Mapping, Peptide,Peptide Fingerprint,Peptide Fingerprints,Protein Fingerprint,Protein Fingerprints
D011994 Recombinant Proteins Proteins prepared by recombinant DNA technology. Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D002853 Chromatography, Liquid Chromatographic techniques in which the mobile phase is a liquid. Liquid Chromatography
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D006868 Hydrolysis The process of cleaving a chemical compound by the addition of a molecule of water.
D000595 Amino Acid Sequence The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION. Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D013056 Spectrophotometry, Ultraviolet Determination of the spectra of ultraviolet absorption by specific molecules in gases or liquids, for example Cl2, SO2, NO2, CS2, ozone, mercury vapor, and various unsaturated compounds. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed) Ultraviolet Spectrophotometry

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