AUTOMATED PROTEIN SEQUENCING BY MICRO-LC/ELECTROSPRAY MS

THE OVERALL GOAL OF THIS PROJECT IS TO DEVELOP A HIGH-SPEED AUTOMATED PROTEIN SEQUENCER EMPLOYING HIGH-PERFORMANCE PACKED CAPILLARY LC SEPARATION, IMMOBILIZED ENZYMES, AND ELECTROSPRAY MASS SPECTROMETRY. THIS SYSTEM SHOULD PROVIDE MOLECULAR WEIGHT AND SEQUENCE INFORMATION USING SAMPLE AMOUNTS IN THE 1-10 PMOL LEVEL AND COMPLETE SEQUENCES AT THE 10-100 PMOL LEVEL. THIS NEW SEQUENCER SHOULD BE TECHNICALLY AND ECONOMICALLY COMPETITIVE WITH THE MODERN GAS PHASE EDMAN SEQUENCER, BUT ORDERS OF MAGNITUDE FASTER, AND SOMEWHAT MORE RELIABLE AND VERSATILE IN THAT IT SHOULD BE ABLE TO DEAL WITH BLOCKED N-TERMINI AND UNUSUAL AMINO ACID RESIDUES. THE APPROACH IS BASED ON EARLIER SUCCESSFUL WORK USING STANDARD-BORE COLUMNS WITH THERMOSPRAY MASS SPECTROMETRY, BUT TAKES ADVANTAGE OF THE MUCH HIGHER SENSITIVITIES WHICH ARE POSSIBLE WITH PACKED CAPILLARY LC INTERFACED TO ELECTROSPRAY MS. AN IMPORTANT NEW COMPONENT TO THE APPROACH IS THE COMBINATION OF BOTH AMINOPEPTIDASES AND CARBOXYPEPTIDASES WITH COLLISION INDUCED DISSOCIATION OF DOUBLY CHARGED TRYPTIC PEPTIDES TO PROVIDE COMPLETE SEQUENCES ON LOW PICOMOLE QUANTITIES OF TRYPTIC FRAGMENTS PRODUCED AND ISOLATED ON-LINE. IN PHASE I, IT WILL BE ESTABLISHED THAT THE APPROACH GIVES RELIABLE, UNAMBIGUOUS SEQUENCE INFORMATION, AND IT WILL BE SHOWN THAT THE SENSITIVITIES ARE NOW FEASIBLE WITHIN THE PRESENT PERFORMANCE OF COMMERCIAL ELECTROSPRAY MASS SPECTROMETERS, FURTHER IMPROVEMENT IN NEW MASS SPECTROMETRIC TECHNIQUES APPLICABLE TO PROTEINS AND PEPTIDES ARE CONSIDERED LIKELY, AND AS THE SENSITIVITY OF THE MS IMPROVES SO WILL THE SENSITIVITY OF THIS NEW APPROACH TO SEQUENCING.