Anal. a m . 1001, 63, 1091-1097
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Primary Structure Determination of Peptides and Enzymatically Digested Proteins Using Capillary Liquid Chromatography/Mass Spectrometry and Rapid Linked-Scan Techniques D. B. Kassel,*f**B. D. Musselman,’ and J. A. Smithla Departments of Pathology and Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, and JEOL USA, Inc., Peabody, Massachusetts 01960
Primary protein seqwncw w e r e d e t ~ f o both r peptM08 and enzymatically digested protelnr by rapid ilnked-scan ( B / E ) liquid chromatography/mass spectrometry/macls spectrometry (LC/MS/MS) at the bw-pimmb k v d (1040 pmd). Rnhg the cowso of a skrgk LC/MS/MS anatysk, we d o ” t r a t e d that it Is pordblo to generate Interpretable dlmodakn spectra of the dutlng protedytlc poptkles. Molecular weights of tryptk peptides were estabI W by using ’Ilo of the proteln d w s t by operating In the caplllary LC/W-FABMS mode. Peptides exhibiting the strongest MH+ Ions were then selected for wbsequent LC/ W/MS analysk (typicah ‘I6 af the mdnhg protein dlge61). Elution times for each chromatographk peak were generally >30 8. I t was theretore possbie to obtdn a mini" of dx B / E fast linked-scan spectra during the course of elution of each peptide component. Typically, B/€ linked scans of the greatesl Ion abundance (obtained at the chromatographic peak maxl”) were averaged to enhance the dgnaWnolse ratio at these low-plcomoie levels. Unit resolution was observed for product Ions below m / r 1000. Rapid linked scanning by LC/frit-FABMSIMS provided mass asslgments for product Ions wlthln 0.2-0.3 amu of theoretical values. Sld.chain fragment ions (w, and d,) were also observed, whkh ahwed for the differentiation of lsobarlc amino acids (e.g., kudrn and boieuch). Exampkr of the appllcatkn of thk fast Unkodaan technique to LC/MS/MS are presented for complex mlxtures of unknown peptldes and the tryptlc digestion of phosphorylated@casein.
INTRODUCTION The mapping of enzymatically digested proteins and the structural characterization of the peptide components is facilitated by directly coupling liquid Chromatography and mass spectrometry (LC/MS) and by LC/MS/MS. A tremendous sensitivity advantage is made when operating the mass spectrometer in this mode. Continuous-flow FAB, developed by Ito et al. (I)and Caprioli et al. (2,3),produces a several-fold detection enhancement over static FAB, which is attributed mainly to the optimization of analyte to matrix ratio at the tip of the FAB probe. High-performance liquid chromatography (HPLC) utilizing gradient, rather than isocratic, elution has the added feature of allowing dilute protein solutions to be concentrated at the head of the HPLC column prior to their *To whom correspondence should be addressed at Glaxo Inc., Five Moore Dr, Research Triangle Park, NC 27709. Department of Pathology, Massachusetts General Hospital. *Department of Molecular Biology, Massachusetts General Hospital. Harvard Medical School. JEOL USA.
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separation. The ability to concentrate dilute solutions prior to gradient elution and the ability to separate complex mixtures in time make LC/MS an ideal method for handling dilute solutions of synthetic peptides and enzymatically digested proteins. A more significant reason, perhaps, is that the quantity of purified protein available to the mass spectrometrist for characterization is often small (200 pmol) separated chromatographically on 1.0-mm microbore LC columns could be ionized directly in an ion-spray source and subsequently collisionally dissociated to produce interpretable product ion spectra. Several others (8-11) have demonstrated the potential of electrospray MS/MS for sequencing tryptic peptides albeit with some difficulty in assigning primary protein sequences for unknowns. Packed-capillary liquid chromatography offers the ability to couple HPLC with mass spectrometry directly without the requirement for postcolumn splitting because of the low flow rates used (2-5 pljmin). Cappiello et al. (12) and Henzel et al. (13)demonstrated that packed capillary columns could be readily coupled with mass spectrometry by using a frit-FAB LC/MS interface (1). The latter used the technique to generate tryptic maps of some genetically engineered, overexpressed proteins at the 5-50-pmol level (14). FAB experiments that employ slurry-packed fused-silica capillary HPLC columns and the postcolumn addition of FAB matrix for their optimum performance have also been reported (15, 16). The capacity for sequencing proteolytic peptides by capillary LC/MS/MS has been demonstrated previously. Cappiello et al. (12) applied a four-sector mass spectrometer equipped with optical array detection to the characterization of hemoglobin variants. In this study, LC effluent was either analyzed directly or, in cases where peptides had overlapping retention times, stored into small loops and subsequently bled into the mass spectrometer at low flow rates and the CID spectra were recorded. The array detector was used to “capture” a CID spectrum on approximately 200 pmol of a tryptic peptide eluting from a capillary HPLC column. Tomer
0003-2700/@1/0363-1091$02.50/0 0 1991 Amerlcan Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 83, NO. 11, JUNE 1, 1991
and co-workers reported collision-induced dissociation (CID) spectra obtained on-the-fly by capillary zone electrophoresis/mass spectrometry/mass spectrometry (CZE/MS/MS) (17)and coaxial continuous-flow FABMS/MS (18) for the analysis of tripeptides and some higher mass peptides (e.g., Bradykinin). Concentrations reported in the latter study related to the amount of sample consumed (i.e., the amount of analyte solution injected) during the analysis and did not reflect directly the amount of starting peptide solution needed. These separation systems required very small injection volumes (typically
