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Current chromatographic and mass spectrometric techniques have limitations for analyzing heparin and heparin oligomers due to their high polarity, structural diversity, and sulfate lability. A rapid method for the analysis of heparin oligosaccharides was developed using ion-pair reversed-phase ultraperformance liquid chromatography coupled with electrospray quadruple time-of-flight mass spectrometry (IPRP-UPLC ESI Q-TOF MS). The method utilizes an optimized buffer system containing a linear pentylamine and a unique additive, 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), to achieve highly efficient separation together with enhanced mass response of heparin oligosaccharides. Analyses of a heparin oligosaccharide test mixture, dp6 through dp22, reveal that the chromatographic conditions enable baseline resolution of isomeric heparin oligosaccharides (dp6) and produce intact molecular ions with no sulfate losses during mass spectrometric analysis. In addition, the described conditions are amenable to the detection of heparin oligosaccharides in positive ion mode, yield stronger positive ion signals for corresponding oligosaccharides compared to the negative ion mode, and allow identification of structural isomers by an MS/MS approach. Because sensitive detection of oligosaccharides is also achieved with ultraviolet (UV) detection, the method utilizes a dual detection scheme (UV and MS in series) along with IPRP UPLC to simultaneously obtain quantification (UV) and characterization (MS) data for heparin oligosaccharides. The broad potential of this new method is further demonstrated for the analysis of a low-molecular-weight heparin (LMWH) preparation from porcine heparin. This approach will be of particular utility for profiling the molecular entities of heparin materials, as well as for structural variability comparison for samples from various sources.

An improved method for peptide de novo sequencing by MALDI mass spectrometry is presented. The method couples a charge derivatization reaction with C-terminal digestion to modify tryptic peptides. The charge derivatization attaches a fixed charge group onto the N-termini of peptides, and the enzymatic digestion after the derivatization step removes C-terminal basic amino acid residues such as arginine and lysine. The fragmentation of the modified peptide(s) under low-energy CID conditions (MALDI Q-TOF mass spectrometer) yields a simplified yet complete ion series of the peptide sequence. The validity of the method is demonstrated by the results from several model protein digests, where peptide sequences were correctly deduced either manually or through an automated sequencing program.

A pair of isotopically coded light and heavy reagents, tris(2,4,6-trimethoxyphenyl)phosphonium acetic acid N-hydroxysuccinimide esters (1 and 2) were synthesized and used to derivatize low molecular weight (