gamma-Carboxyglutamic acids 36 and 40 do not contribute to human factor IX function.

The gamma-carboxyglutamic acid (Gla) domains of the vitamin K-dependent blood coagulation proteins contain 10 highly conserved Gla residues within the first 33 residues, but factor IX is unique in possessing 2 additional Gla residues at positions 36 and 40. To determine their importance, factor IX species lacking these Gla residues were isolated from heterologously expressed human factor IX. Using ion-exchange chromatography, peptide mapping, mass spectrometry, and N-terminal sequencing, we have purified and identified two partially carboxylated recombinant factor IX species; factor IX/gamma 40E is uncarboxylated at residue 40 and factor IX/gamma 36,40E is uncarboxylated at both residues 36 and 40. These species were compared with the fully gamma-carboxylated recombinant factor IX, unfractionated recombinant factor IX, and plasma-derived factor IX. As monitored by anti-factor IX:Ca (II)-specific antibodies and by the quenching of intrinsic fluorescence, all these factor IX species underwent the Ca(II)-induced conformational transition required for phospholipid membrane binding and bound equivalently to phospholipid vesicles composed of phosphatidylserine, phosphatidylcholine, and phosphatidylethanolamine. Endothelial cell binding was also similar in all species, with half-maximal inhibition of the binding of 125I-labeled plasma-derived factor IX at concentrations of 2-6 nM. Functionally, factor IX/gamma 36,40E and factor IX/gamma 40E were similar to fully gamma-carboxylated recombinant factor IX and plasma-derived factor IX in their coagulant activity and in their ability to participate in the activation of factor X in the tenase complex both with synthetic phospholipid vesicles and activated platelets. However, Gla 36 and Gla 40 represent part of the epitope targeted by anti-factor IX:Mg(II)-specific antibodies because these antibodies bound factor IX preferentially to factor IX/gamma 36,40E and factor IX/gamma 40E. These results demonstrate that the gamma-carboxylation of glutamic acid residues 36 and 40 in human factor IX is not required for any function of factor IX examined.

Identification of the facile gas-phase cleavage of the Asp-Pro and Asp-Xxx peptide bonds in matrix-assisted laser desorption time-of-flight mass spectrometry.

Abundant ions corresponding to the gas-phase cleavage of the Asp-Pro and Asp-Xxx bonds of peptides in the process of matrix-assisted laser desorption were observed using a time-of-flight mass spectrometer equipped with both linear and reflector mass analyzers. Peptides containing the N-terminal sequence, Asp-Pro ... from an endoproteinase Asp-N digest yielded one peak in the molecular ion region in the linear mode and two equally abundant peaks in the reflector mode TOF mass spectra. The lower molecular masses in the reflector mode mass spectra could be eliminated by removing the Asp residue or derivatizing its side-chain carboxyl group. The observed mass differences did not correspond to any amino acid; however, by lowering the potential of the reflector to correct for the energy loss the mass difference was determined to be 115 Da, i.e., Asp. The extent and rate of this decomposition was compared with that obtained using a four-sector tandem mass spectrometer in the MS/MS mode of operation without and with a collision gas at collision cell potentials of 3.0 and 9.86 kV. These data suggest the Asp-Pro peptide bond is more labile than other peptide bonds in the gas phase. Abundant metastable decomposition of internal Asp-Pro bonds was also observed in larger peptides and proteins. Based on these latter data, a mechanism for this gas-phase cleavage is proposed.(ABSTRACT TRUNCATED AT 250 WORDS)

Site-specific carbohydrate identification in recombinant proteins using MALD-TOF MS.

The utility of matrix-assisted laser desorption time-of-flight (MALD-TOF) mass spectrometry for the analysis of recombinant glycopeptides is discussed and compared to information which may be obtained by fast atom bombardment mass spectrometry (FABMS) and tandem mass spectrometry (MS/MS). MALD-TOF appears to be 10-100 times more sensitive than FAB MS for the analysis of underivatized glycopeptides, providing qualitative site-specific information regarding the carbohydrate microheterogeneity without the extensive isolation and derivatization procedures required to obtain similar information by FAB MS. Analysis of a digest mixture in the positive and negative ion mode of MALD-TOF indicated that, in mixtures, sialylated glycopeptides are preferentially detected in the negative ion mode. The determination of the molecular masses of a glycopeptide with MALD-TOF prior to and after treatment with a variety of specific glycosidases, often without removal of the buffers, coupled to a comparison of molecular mass information available from a carbohydrate database facilitates the assignment of a carbohydrate composition. The vast majority of the molecular ion signal observed in the linear mode for sialylated glycopeptides are metastable ions. Reflector mass spectra reveal a shift to lower mass consistent with the loss of most of the neuraminic acid residues. The loss of Hex and HexNAc residues is also observed. Sequential lowering of the reflector potential reveals structurally significant fragment ions representing the carbohydrate and peptide portions of the molecule.