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

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.