Purification and characterization by fast-atom-bombardment mass spectrometry of the polymorphonuclear-leucocyte-elastase-generated A alpha (1-21) fragment of fibrinogen from human blood after incubation with calcium ionophore A23187.
The stimulation of human blood with a Ca2+ ionophore, A23187, leads to activation of polymorphonuclear leucocytes (PMN) with release of small amounts of catalyticaly active elastase, as demonstrated by the formation of a characteristic product, the N-terminal A alpha (1-21) peptide of the Aa subunit of fibrinogen. The identity of the peptide was initially established by radioimmunoassay (r.i.a.) with an antibody raised to A alpha (1-21). We now provide independent confirmation of the formation of A alpha (1-21) by fast-atom-bombardment-m.s. analysis of the fractions separated chromatographically after spiking of plasma samples with peptide labelled with [2H8]Phe at position 8. Identity of the peptides was established on the basis of their chromatographic retention time and by the distinct peaks in the mass spectra of these fractions. The relative intensities of the molecular ions of natural and labelled peptides were measured. On the basis of a comparison of the peaks of similar intensities, the concentration of the natural peptide at the time of spiking was close (79%) to the amount obtained by r.i.a. An additional peptide, des-alanyl-A alpha (2-21), was also seen. The total amount of material measured by r.i.a. could be accounted for by the sum of these two provides. The addition of label and assay by m.s. has provided an independent physical-chemical method for identifying A alpha (1-21) as a characteristic product of PMN elastase release in whole blood, but which is absent in freshly drawn blood.
Dewey
R S
RS
Merck Sharp and Dohme Research Laboratories, Rahway, NJ 07065.
Liesch
J M
JM
Williams
H R
HR
Sugg
E E
EE
Dolan
C A
CA
Davies
P
P
Mumford
R A
RA
Albers-Schönberg
G
G
D000595:Amino Acid Sequence; D000001:Calcimycin / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D005340:Fibrinogen / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology*; D010196:Pancreatic Elastase / Q000097:blood*; D010446:Peptide Fragments / Q000097:blood; D016339:Spectrometry, Mass, Fast Atom Bombardment
D010446:Peptide Fragments; D000001:Calcimycin; D005340:Fibrinogen; D010196:Pancreatic Elastase