Mass Spectrometry-Based Glycoproteomic Approach Involving Lysine Derivatization for Structural Characterization of Recombinant Human Erythropoietin

Abstract

Lysine-containing peptides comprising glycosylation sites derived from recombinant human erythropoietin (rHuEPO) by trypsin or Lys-C and PNGase F dual digestion were derivatized with 2-methoxy-4,5-dihydro-1H-imidazole and its deuterated analogues. In the same reaction, under reducing conditions (beta-mercaptoethanol), cysteines were converted into methyl-cysteines and lysines into Lys-4,5-dihydro-1H-imidazole. Both modifications on cysteines and lysines simplified the CID-MS/MS spectra, while preserving the structural information by yielding y-series ions and improved the mass spectral signal intensity up to 25 times. Moreover, by this approach, the N-glycan occupation sites were unambiguously determined. O-Glycosylation sites as well as O-glycan structures were determined by a LC-MS/MS experiment carried out on dually digested rHuEPO. N-Glycan mixture purified on a graphitized carbon column using a newly developed method that extracted only sialylated carbohydrates was analyzed first using MALDI-TOF in negative linear ion mode with low mass accuracy but without interferences and metastabile ions and then a reflectron with high mass accuracy. After defining the precursor ions, we performed the nanoESI QTOF MS/MS analysis on N-glycans, mainly targeting the distinction between carbohydrates with sialylated antennae and those lacking sialic acid moieties.

Profiling of N‐ and O‐glycopeptides of erythropoietin by capillary zwitterionic type of hydrophilic interaction chromatography/electrospray ionization mass spectrometry

“…Consequently, protease digest of rhEPO may be a good model sample for the present study. Although similar attempts using the RP (C18 and C4)-HPLC have already been reported [12,13], to the best of our knowledge, there has been little investigation of HILIC or ZIC-HILIC separation for rhEPO glycopeptides and other complex mixtures of both N-and O-glycopeptides. In the present work, 105 N-glycopeptides and 8 O-glycopeptides were detected during a single run of the capillary ZIC-HILIC/ESI MS analysis using 150 ng rhEPO.…”

“…Recently, several new methods for the purification/ enrichment of glycopeptides (e. g., chemical trapping [7,8], sepharose adsorption [9], and SPE using porous graphite carbon (PGC) [10] or zwitterionic-type hydrophilic interaction chromatography (hereafter called ZIC-HILIC) [11]) have been developed. Selective separation methods of glycopeptides using RP [12,13] and HILIC [14 -16] columns have also been reported. Very recently, we reported that a ZIC-HILIC column with sulfobetaine groups [17] has a high capability for separation based on its structural recognition of N-glycans and selectivity for N-glycopeptides [18,19].…”

Inductive properties of polypyridyl ruthenium complexes significantly regulate various protein distributions in Escherichia coli

“…Preparation of tryptic peptide fragments [26,30] The desired bands were excised from the SDS-polyacrylamide gel or 2D gel, and dried by the sequential procedures of destaining with (NH 4 )HCO 3 (50 mM in 50% [v/v] CH 3 CN) to remove the Coomassie Brilliant Blue, dehydration with CH 3 CN (50 μL), and drying with a SpeedVac to produce a white solid. A reducing reagent (10 mM DTT in 50 mM (NH 4 )HCO 3 ) was then added to the gel slices at 50°C for 60 min.…”

Electrospray ionization mass spectrometry as a critical tool for revealing new properties of snake venom phospholipase A2

“…Then 1.3 µL of Triton X‐100 and 1.0 µL of PNGase F were added to the reaction. Proteolysis time lasted for 3 h at 37°C 27, 28…”