CAPILLARY LIQUID-CHROMATOGRAPHY COUPLED WITH AN ION-TRAP STORAGE REFLECTRON TIME-OF-FLIGHT MASS-SPECTROMETER FOR STRUCTURAL CONFIRMATION OF3 RECOMBINANT PROTEIN ISOFORMS

Packed-capillary high-performance liquid chromatography (HPLC) was suc cessfully coupled with an ion trap storage/reflectron time-of-flight m ass spectrometer (LC/IT/reTOFMS) through an electrospray ionization in terface for protein structural elucidation. Using the total-ion storag e capabilities of the trap over a broad mass range and the high sensit ivity from the packed capillary column with i.d. as small as 250 mu m, high sensitivity peptide mapping in the low picomole range was demons trated for the structural confirmation of three recombinant human nucl eoside diphosphate kinase isoforms (NDPK, E.C. 2.7.4.6). A strategy co mbining chemical/enzymatic digestions as well as collisionally-induced dissociation (CID) in the electrospray source was successfully employ ed to infer the minor primary structural differences among the three r ecombinant proteins. This high sensitivity was achieved while also mai ntaining a resolution of nearly 1500 for mass identification using the capabilities of the lT/reTOF device. A point mutation of serine 120 t o glycine was verified between the wildtype NDPK A and its mutant (Del ta m=30 u) by both selected-ion monitoring and ion-source CID of the p rotein fragment containing the mutation site. For the structural confi rmation of the sequence of NDPK A and B (88% homology), two sets of ch emical/proteolytic digests were generated independently and followed b y LC/MS analysis of the molecular weight of each protein-generated fra gment. The complementary information from the two chromatographic anal yses allowed for sequence verification of the two protein isoforms. Th e experiments clearly demonstrated that the high concentration sensiti vity of the capillary high-performance liquid chromatographic separati on together with the advantages of the IT/reTOF mass spectrometer coul d provide a low-cost, high-performance facility for protein analysis.