Matrix‐assisted laser desorption/ionization on‐target method for the investigation of oligosaccharides and glycosylation sites in glycopeptides and glycoproteins

Abstract

The significance of glycoproteins in living systems instigates the ceaseless expansion of new techniques and procedures for the analysis of biological samples. Many of these applications are focused on improving the detection limit of analyzed material. In a previous study, we described a procedure for the detection of oligosaccharides cleaved from tryptic glycopeptides. Treatment of deglycosylated fractions with phenylhydrazine gave rise to peaks consistent with labeled glycans, and both types of compounds--deglycosylated peptides and oligosaccharides--were recorded from one spot and observed in one matrix-assisted laser desorption/ionization (MALDI) mass spectrum for the first time. Here, we added an additional step to this simple procedure of deglycosylating glycopeptides directly from the target spot of the first analyzed glycosylated peptides. For the purpose of this new study, a mixture of 2-aza-2-thiothymine and phenylhydrazine hydrochloride showed to be an excellent matrix for glycopeptides, oligosaccharides, deglycosylated peptides and moreover it allowed PNGaseF to be active enough to cleave oligosaccharides from peptides. The efficiency of this procedure is demonstrated on a series of intact glycoproteins and on the analysis of tryptic peptides obtained from IgG and total mouse serum. This one-step on-target deglycosylation method with subsequent derivatization on the same spot makes MALDI-MS analyses of glycopeptides fast, simple and accessible for biological samples, where classical procedures cannot produce useful results.

Ex vivo assays of CEM cells cultured and treated in the three dimensional cultures

“…Both these ions indicated a peptide with the same mass (m/z 1189.5) but differing in the glycosylation patterns. To confirm the origin of the peptide sequence and the composition of oligosaccharides linked at asparagine, glycans were enzymatically detached with PNGase and analyzed next to the deglycosylated peptides [17] and [18]. The fragmentation pattern of the peptide observed at m/z 1190.5 was consistent with the sequence of amino acids EEQYDSTYR described previously in human IgG [17] and [23].…”

“…MS results were confirmed by sequencing the tryptic peptides in MS/MS mode, and ion peaks assigned manually or searched against the National Centre for Biotechnology Information database Mascot MS/MS system. Deglycosylation of the glycopeptide mixtures was performed with peptide-N-glycosidase F (PNGaseF) treatment according to the procedures described previously [17] and [18].…”

Combined treatment of human MCF-7 breast carcinoma with antibody, cationic lipid and hyaluronic acid using ex vivo assays

“…There is a clinical evidence that trastuzumab, a monoclonal antibody targeting the HER-2 receptor, is an important component of the first-line treatment for patients with metastatic breast cancer. However, the clinical issue of resistance to HER-2 directed therapy still remains unsolved [22]. As a result of the resistance, overexpression of HER-2 has been associated with more aggressive tumor behavior and poorer prognosis.…”

N-Glycomic Changes in Human Breast Carcinoma MCF-7 and T-Lymphoblastoid Cells After Treatment with Herceptin and Herceptin/Lipoplex

“…was mixed by flushing the pipet tip several times and then loaded onto the spot with matrix (DHB or ATT/PHN.HCl in molar ratio 2:1 43 ). The target was left at room temperature until the spots had dried.…”