Capillary electrophoresis-atmospheric pressure ionization mass spectrometry for the characterization of peptides. Instrumental considerations for mass spectrometric detection.
On-line capillary electrophoresis (CE) separations are shown for a synthetic peptide mixture and a tryptic digest of human hemoglobin in an uncoated fused-silica capillary with detection using atmospheric pressure ionization mass spectrometry (API-MS). The CE system utilized a 1-m capillary column of either 75- or 100-microns I.D. These somewhat larger inside diameters allow higher sample capacities for MS detection and the 1-m length facilitates connecting the CE column to the liquid junction-ion spray interface and MS system. Low volatile buffer concentrations (15-20 mM) of ammonium acetate or ammonium formate, and high organic modifier content (5-50%) of methanol or acetonitrile facilitates ionization under electrospray conditions. This study shows that peptides separated by CE may be transferred to the API-MS system through a liquid junction coupling to the pneumatically assisted electrospray (ion spray) interface at low buffer pH when the electroosmotic flow is low (0-0.04 microliter/min). CE-MS as described herein is facilitated by features in modern CE instrumentation including robotic cleaning and pressurization of the capillary inlet. The latter is particularly useful for repetitive rinsing and conditioning of the capillary column between analyses in addition to continuous 'infusion' of sample to the mass spectrometer for tuning purposes. In addition to facile molecular weight determination, amino acid sequence information for peptides may be obtained by utilizing on-line tandem MS. After the tryptic digest sample components enter the API-MS system, the molecular ion species of individual peptides may be focussed and transmitted into the collision cell of the tandem triple quadrupole mass spectrometer. Collision-induced dissociation of protonated peptide molecules yielded structural information for their characterization following injection of 10 pmol of a tryptic digest from human hemoglobin.
Johansson
I M
IM
Drug Testing and Toxicology, Cornell University, Ithaca, NY 14850.
Huang
E C
EC
Henion
J D
JD
Zweigenbaum
J
J
D000097:Acetonitriles; D002197:Capillary Action; D004586:Electrophoresis / Q000379:methods*; D006454:Hemoglobins / Q000302:isolation & purification; D006801:Humans; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry / Q000379:methods*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification*; D014357:Trypsin
D000097:Acetonitriles; D006454:Hemoglobins; D010446:Peptide Fragments; D010455:Peptides; D014357:Trypsin; C032159:acetonitrile