No abstract

Electrospray ionization mass spectrometry (ESI-MS) is a valuable tool in structural biology for investigating globular proteins and their biomolecular interactions. During the electrospray ionization process, proteins become desolvated and multiply charged, which may influence their structure. Reducing the net charge obtained during the electrospray process may be relevant for studying globular proteins. In this report we demonstrate the effect of a series of inorganic and organic gas-phase bases on the number of charges that proteins and protein complexes attain. Solution additives with very strong gas-phase basicities (GB) were identified among the so-called "proton sponges". The gas-phase proton affinities (PA) of the compounds that were added to the aqueous protein solutions ranged from 700 to 1050 kJ mol(-1). Circular dichroism studies showed that in these solutions the proteins retain their globular structures. The size of the proteins investigated ranged from the 14.3 kDa lysozyme up to the 800 kDa tetradecameric chaperone complex GroEL. Decharging of the proteins in the electrospray process by up to 60 % could be achieved by adding the most basic compounds rather than the more commonly used ammonium acetate additive. This decharging process probably results from proton competition events between the multiply protonated protein ions and the basic additives just prior to the final desolvation. We hypothesize that such globular protein species, which attain relatively few charges during the ionization event, obtain a gas-phase structure that more closely resembles their solution-phase structure. Thus, these basic additives can be useful in the study of the biologically relevant properties of globular proteins by using mass spectrometry.

The recently introduced electron transfer dissociation (ETD) technique opens new possibilities for the structural characterization of glycoproteins at the glycopeptide level. In this report, we investigate the ETD mass spectra of tryptic N-glycopeptides of the model glycoprotein horseradish peroxidase (HRP). Multiply protonated N-glycopeptides obtained by electrospray ionization were subjected to ETD. Fragment ions obtained by ETD were further analyzed by collision-induced dissociation (CID) (MS(3)) for their unambiguous structural assignment. The following fragmentation features were revealed: (1) c- and z-type peptide backbone cleavages were observed with retention of the intact glycan moiety revealing peptide sequence, glycan attachment site, and glycan mass; (2) to a lesser extent, glycosidic bond cleavages were registered with retention of the intact peptide sequence; and (3) a range of amino acid side chain losses did occur. Remarkably, the loss of the complete N-glycosylated asparagine side chain was observed. This loss of the glycan-modified side chain helps with the structural characterization of glycopeptides by allowing the facile deduction and verification of the glycan mass and the nature of the amino acid residue at the glycan attachment site. Importantly, informative ETD spectra were obtained in this study by reversed-phase nano-liquid chromatography (LC) coupled online to a radio-frequency (rf) quadrupole ion trap (QIT) mass spectrometer with alternating acquisition of CID and ETD mass spectra from an automatically selected set of precursors (data-dependent mode). Thus, our study brings nano-LC/QIT-MS(n) with CID and ETD to the fore as a powerful technique for glycoproteomics at the glycopeptide level.

Schistosomes are parasitic flatworms that infect millions of people in (sub)tropical areas around the world. Glycoconjugates of schistosomes play a critical role in the interaction of the different developmental stages of the parasite with the host. In particular, glycosylated components of the eggs produced by the adult worm pairs living in the bloodstream are strongly immunogenic. We have investigated the glycosylation of interleukin‐4‐inducing factor from schistosome eggs (IPSE/alpha‐1), a major secretory egg antigen from Schistosoma mansoni that triggers interleukin‐4 production in human basophils, by MS analysis of tryptic glycopeptides. Nanoscale LC‐MS(/MS) and MALDI‐TOF(/TOF)‐MS studies combined with enzymatic degradations showed that monomeric IPSE/alpha‐1 contains two N‐glycosylation sites, which are each occupied for a large proportion with core‐difucosylated diantennary glycans that carry one or more Lewis X motifs. Lewis X has been reported as a major immunogenic glycan element of schistosomes. This is the first report both on the expression of Lewis X on a specific schistosome egg protein and on a protein‐specific glycosylation analysis of schistosome eggs.

No abstract