Glycoprotein profiling by electrospray mass spectrometry

Abstract

This work compares several different methods of site-specific analysis of glycoproteins using electrospray mass spectrometry. The glycoprotein, oLH␣ (ovine luteinizing hormone, ␣-subunit) was chosen as an appropriate example protein for these studies because of its biological relevance and extreme microheterogeneity. More than 20 unique glycoforms were detected for this glycoprotein at the Asn 56 site of oLH␣. The carbohydrates present at this site affect receptor binding affinity, so understanding the great variety in the composition of these carbohydrates is important in studying ligand binding interactions. MS data was acquired on a quadrupole ion trap, a triple quadrupole, and a quadrupole time of flight mass spectrometer, and carbohydrate composition at the Asn 56 site of oLH␣ was determined using these instruments. Additionally, neutral loss and precursor ion scanning modes were also used to identify the glycoforms present, and these techniques were compared to the standard MS data. Of the three instruments compared in the study, the qTOF mass spectrometer achieved the lowest sample consumption, but all three instruments were useful in profiling the glycopeptide composition. (J Am Soc Mass Spectrom 2004, 15, 750 -758)

A novel approach for identification and characterization of glycoproteins using a hybrid linear ion trap/FT-ICR mass spectrometer

“…Generating diagnostic oxonium ions from glycopeptides in the skimmer region of a mass spectrometer has been well described in the literature [5,10,14,27]. In brief, a high potential is placed on the ion optics following the skimmer, such that ions are accelerated in a region that has moderate pressure, causing ion fragmentation.…”

Enhanced detection of sulfated glycosylation sites in glycoproteins

“…Glycopeptides from ovine luteinizing hormone, ␣ subunit, were generated in the laboratory of Dr. George Bousfield, Wichita State University, as described previously [16]. Briefly, after proteinase K digestion the released glycopeptides were isolated using centrifugal ultrafiltration and desalted.…”

“…The glycosylation was quite variable; it included sulfated and nonsulfated glycoforms. In all, about 20 different glycoforms were present in the mixture, based on mass spectrometric analysis [16]. For ion-pairing experiments, this sample, referred to henceforth as "the sulfated glycopeptide mixture", was combined with the ion-pairing reagent, Lys-LysLys.…”

“…Enzymatic digestion of glycoproteins, followed by mass spectral analysis of the resulting glycopeptide mixtures, has been implemented as an effective strategy to profile glycosylation in glycoproteins [16,[23][24][25]. (Both tryptic digests and digests using nonselective enzymes like proteinase K have been used for these purposes.)…”

“…In addition to the problems of detecting glycopeptides in negative ESI, it is well known that in positive ion mode the signal of sulfated compounds is suppressed by mass spectral signals of nonsulfated compounds. We have demonstrated this recently in the analysis of sulfated glycopeptides released from luteinizing hormone [16]. In essence, the detection of (underivatized) sulfated glycopeptides is not ideal in negative mode or positive mode, and complementary detection methods are required to improve the detection and characterization of these compounds.…”