Improved tryptic digestion assisted with an acid-labile anionic surfactant for the separation and characterization of glycopeptide glycoforms of a proteolytic-resistant glycoprotein by capillary electrophoresis time-of-flight mass spectrometry

Abstract

Certain glycoproteins are rather difficult to digest due to compacted tertiary or quaternary structures. In a previous study, a capillary LC coupled to TOF-MS (μLC-TOF-MS) method was developed for the detection and characterization of the glycopeptide glycoforms of human transferrin (Tf), a proteolytic resistant glycoprotein, in serum samples. After immunoaffinity purification, Tf was digested with trypsin in the presence of RapiGest(®) and μLC-TOF-MS analyses permitted to detect the N413 and N611 glycopeptide glycoforms. Conversely, the use of this surfactant, albeit mandatory to quantitatively digest the isolated Tf, proved detrimental to CE-TOF-MS analysis due to its interaction with the inner surface of the silica capillary walls. As CE is usually regarded as an interesting alternative to other separation techniques (low consumption of reagents, excellent separation efficiency, and reduced analysis times), in this work, the undesirable interferences of the surfactant have been removed to allow the correct separation and detection of Tf glycoforms by CE-TOF-MS. Moreover, the digestion protocol described by the RapiGest(®) manufacturer has been modified to minimize desialylation of Tf glycopeptide glycoforms. The new developed CE-TOF-MS methodology has been then compared with the former μLC-TOF-MS by means of sensitivity and separation efficiency of Tf glycopeptide glycoforms in the standard glycoprotein. Additionally, Tf glycopeptide glycoforms from serum of healthy volunteers and patients with congenital disorders of glycosylation have also been analyzed following the developed methodology.

Recent advances in mass spectrometric analysis of glycoproteins

“…To improve the separation and detection of human transferrin glycoforms by CE‐TOF‐MS, a modified method was developed by eliminating undesirable interferences, caused by surfactant, with a desalting procedure using μElution plates. Suppression of the desialylation of glycopeptide glycoforms was also achieved using 5% of HF, for 30 min at 37ºC, as the optimum conditions to hydrolyze residual surfactant . The methodology was further confirmed following the successful analysis of human transferrin glycopeptides in serum samples from a healthy control and patients with congenital disorders of glycosylation.…”

Recent developments in capillary and microchip electroseparations of peptides (2015–mid 2017)

“…Improved tryptic digestion assisted with an acid‐labile anionic surfactant for the separation and characterization of glycopeptide glycoforms of a proteolytic resistant glycoproteins (human transferrin) was monitored by CE‐ESI‐TOF‐MS methodology . The CE separation of glycopeptides were performed in an Agilent 7100 CE apparatus in 70 cm long, 75 μm id bare FS capillary at 18 kV in the BGE containing 50 mM acetic acid and 50 mM formic acid.…”

Capillary Electrophoresis: Trends and Recent Advances

“…Whereas the surfactants are required to ensure full digestion, their elimination at low pH (when using RapiGest) has shown to lead to loss of sialic acid residues on glycan structures of transferrin and distortion of glycopeptide peaks in general. 145 Changing the type of acid and sample desalting helped minimizing desialylation and excellent peak shapes were obtained. CE enabled fast and efficient separation of the sialylated glycopeptides improving detection sensitivity due to decreased ion suppression.…”

“…Surfactants often present a source of undesirable interferences during (glyco)­proteomics studies. Whereas the surfactants are required to ensure full digestion, their elimination at low pH (when using RapiGest) has shown to lead to loss of sialic acid residues on glycan structures of transferrin and distortion of glycopeptide peaks in general . Changing the type of acid and sample desalting helped minimizing desialylation and excellent peak shapes were obtained.…”