Mass spectrometric techniques combined with enzymatic digestions were applied to determine the glycosylation profiles of cellobiohydrolase (CBH II) and endoglucanases (EG I, II) purified from filamentous fungus Trichoderma reesei. Electrospray mass spectrometry (ESMS) analyses of the intact cellulases revealed the microheterogeneity in glycosylation where glycoforms were spaced by hexose units. These analyses indicated that glycosylation accounted for 12-24% of the molecular mass and that microheterogeneity in both N- and O-linked glycans was observed for each glycoprotein. The identification of N-linked attachment sites was carried out by MALDI-TOF and capillary liquid chromatography-ESMS analyses of tryptic digests from each purified cellulase component with and without PNGase F incubation. Potential tryptic glycopeptide candidates were first detected by stepped orifice-voltage scanning and the glycan structure and attachment site were confirmed by tandem mass spectrometry. For purified CBH II, 74% of glycans found on Asn310 were high mannose, predominantly Hex(7-9)GlcNAc(2), whereas the remaining amount was single GlcNAc; Asn289 had 18% single GlcNAc occupancy, and Asn14 remained unoccupied. EG I presented N-linked glycans at two out of the six potential sites. The Asn56 contained a single GlcNAc residue, and Asn182 showed primarily a high-mannose glycan Hex(8)GlcNAc(2) with only 8% being occupied with a single GlcNAc. Finally, EG II presented a single GlcNAc residue at Asn103. It is noteworthy that the presence of a single GlcNAc in all cellulase enzymes investigated and the variability in site occupancy suggest the secretion of an endogenous endo H enzyme in cultures of T. reesei.

中文翻译：

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鉴定里氏木霉的纤维二糖水解酶II和内切葡聚糖酶I和II中的聚糖结构和糖基化位点。

应用质谱技术结合酶消化法确定从丝状真菌里氏木霉纯化的纤维二糖水解酶（CBH II）和内切葡聚糖酶（EG I，II）的糖基化谱。完整纤维素酶的电喷雾质谱（ESMS）分析显示糖基化中的微异质性，其中糖型由己糖单元隔开。这些分析表明糖基化占分子质量的12-24％，并且对于每种糖蛋白，在N-和O-连接的聚糖中均观察到微异质性。通过MALDI-TOF和毛细管液相色谱-ESMS对每种纯化的纤维素酶组分的胰蛋白酶消化物进行分析，并进行PNGase F孵育和不进行PNGase F孵育来鉴定N-连接的附着位点。首先通过逐步孔板电压扫描检测潜在的胰蛋白酶糖肽候选物，并通过串联质谱法确定聚糖结构和附着位点。对于纯化的CBH II，在Asn310上发现74％的聚糖是高甘露糖，主要是Hex（7-9）GlcNAc（2），而其余的是单个GlcNAc。Asn289的单GlcNAc占用率为18％，而Asn14仍未被占用。EG I在六个潜在位点中的两个位点显示了N-连接的聚糖。Asn56包含单个GlcNAc残基，而Asn182主要显示高甘露糖聚糖Hex（8）GlcNAc（2），只有8％被单个GlcNAc占据。最后，EG II在Asn103上显示了单个GlcNAc残基。