Tryptic mapping of human chorionic gonadotropin by matrix‐assisted laser desorption/ionization mass spectrometry

Abstract

The potential of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry for use in the identification of human chorionic gonadotropin (hCG) seized by law enforcement agencies is investigated. Analysis of untreated hCG revealed signals corresponding to the molecular ions of the intact hCG heterodimer and both its non-covalently linked subunits. Unfortunately, due to carbohydrate heterogeneity, the peaks are broad which makes accurate mass assignment, and consequently identification, difficult. Peptide mapping by MALDI-TOF following tryptic digestion gave sequence coverage of 59% and 52% for the alpha- and beta-subunits respectively. Nevertheless, the tryptic map was considered to provide unambiguous identification of hCG. This was confirmed by searching peptide-mass databases with the experimentally determined masses. Our data suggest that peptide mapping by proteolytic digestion followed by MALDI-TOF mass spectrometry is a suitable analytical technique for the identification of hCG seized by the legal authorities.

The beta-subunit of human chorionic gonadotrophin exists as a homodimer

“…Nevertheless, in an earlier study we were able to demonstrate the quaternary structure of the intact hCG heterodimer by MALDI-TOF using an SA matrix (Laidler et al 1995) and in this current investigation we have observed a mass ion akin to that expected of the homodimer in SA and sDHB matrices. Of course, such mass spectral data alone are not unequivocal in showing the presence of the homodimer because proteins may form dimers during ionisation.…”

Mass Spectrometric Characterization of the β-Subunit of Human Chorionic Gonadotropin

“…Interestingly, similar difficulty in detection was described for MALDI-TOF analysis of hCG. 33 Since we were unable to detect native protein or subunit, we focused our attention on the analysis of the b-subunit. The a-and b-subunits of hCG can be readily dissociated with TFA.23 Experiments using a nondenaturing HPLC mobile phase indicated that TFA dissociation of the chains is complete in less than 5 min.…”

Discrimination of mammalian growth hormones by peptide-mass mapping

“…The digests were incubated at room temperature for 24 h. A second aliquot of trypsin (1-2 mL) was added after 4 h to give a final protein to protease ratio of approximately 25 to 1 (w/ w). The eGH formulation was desalted, by centrifugal ultrafiltration using a microconcentrator containing 10 kDa molecular-mass-cutoff membrane (Amicon, Stonehouse, UK) 1 , prior to analysis.…”

“…Nonetheless, peptide-mass mapping may also provide a possible means for end-point detection when developing a method to detect doping with GH. Following identification of human chorionic gonadotropin by peptide-mass mapping, 1 another banned protein hormone in human and equine sports, immunoaffinity extraction was subsequently applied to concentrate this analyte from urine prior to identification by MS. 2 Such an approach may be particularly suited to detect a recent administration of either a cross-species GH or a recombinant GH with an additional methionine residue not present in the endogenous hormone, a distinct possibility with respect to performance dogs and horses. However, peptide-mass mapping is not suitable where a recombinant GH is administered which is identical with that of the species' endogenous GH and in such cases a different approach is required.…”