Enzymes for glycoproteomics
真核生物的 N-聚糖可以使用PNGase F从 Asn 上释放。除非 N-聚糖核心具有在黏菌、植物、昆虫和寄生虫中发现的某些修饰，否则该酶将去除连接到 Asn 上的寡甘露糖型、杂合型和复合型 N-聚糖。
另一种酶称为 PNGase A（来自杏仁），它将去除所有 N-聚糖。这两种酶都是酰胺酶，它们释放连接到 Asn 的氮上的 N-聚糖，从而将 Asn 转化为 Asp。
因此，可以通过在 PNGase F 或 A 处理之前和之后进行的氨基酸序列分析来推断糖基化位点。
其他细菌酶在 N-聚糖核心的两个 GlcNAc 残基之间裂解，留下一个 GlcNAc 连接到 Asn 上。内切糖苷酶 H 释放寡甘露糖型和杂合型 N-聚糖，但不释放复合型 N-聚糖。内切糖苷酶 F1 类似于内切糖苷酶 H，而内切糖苷酶 F2 主要释放双触角 N-聚糖，内切糖苷酶 F3 释放双触角和三触角 N-聚糖，并偏好核心中带有 Fuc 残基的聚糖。
PNGase F
肽N-糖苷酶 F (Peptide -N-Glycosidase F)，即 PNGase F ，是一种酰氨酶，可以从糖蛋白上切除 N-连接型糖链（N-linked glycans）。
切割位置在高甘露糖、杂合和复合寡糖（high mannose, hybrid, and complex）部分最内侧的 GlcNAc 和天冬酰氨残基之间[Anal. Biochem. 1989, 180, 195]。但是，当其最内侧的 GlcNAc残基通过 α1-3 构型连接一个岩藻糖（Fucose）时，PNGase F 则不能切割。
这种
α1-3岩藻糖修饰在植物和部分昆虫来源的糖蛋白中最为常见。换句话说，α1-3 岩藻糖的存在会阻断 PNGase F 的活性，因此在处理非哺乳动物（如植物或昆虫）来源的糖蛋白时，需要特别注意这种结构限制。
对于这类底物，可使用PNGase A。PNGase A 可以切割带有 α1-3 岩藻糖的 N-糖链。
PNGase F 还以不含甘油的形式提供，便于在 HPLC 方法中取得最佳效果。
PNGase F is purified from Flavobacterium meningosepticum (3).
从脑膜败血性黄杆菌（ Flavobacterium meningosepticum ）中提取纯化（ 2 ）。
产品
| 反应条件于糖蛋白变性缓冲液（含 5% SDS ， 400mM DTT ）中 100 ℃ 煮沸 10 分钟使糖蛋白变性。再在加入 1%NP-40 的 1X G7 反应缓冲液 [50 mM 磷酸钠（ pH 7.5@25 ℃ ） ] 中于 37 ℃ 温育，以进行酶切反应。 |
| --- |
| 随酶提供的试剂10X 糖蛋白变性缓冲液 |
10X G7 缓冲液
10% NP-40 |
| 比活性~1,800,000 units/mg 。 |
| |
| 品质保证无外切糖苷酶污染，无内切糖苷酶 F1 、 F2 和 F3 活性。无污染的蛋白水解活性。 |
| 单位定义1 单位指在 10 μl 的反应体系中， 37 ℃ 条件下 1 小时 从 10 μg 变性 RNase B 中除去超过 95% 的碳水化合物所需要的酶量（ 65 NEB units = 1 IUB milliunit ）。 |
| 浓度500,000 units/ml 。 |
| 贮存条件PNGase F ： 50 mM NaCl ， 20 mM Tris-HCl （ pH 7.5@ 25 ℃ ）， 5 mM Na2EDTA 和 50% 甘油。保存于 -20 ℃ 。PNGase F （无甘油型）： 50 mM NaCl ， 20 mM Tris-HCl （ pH7.5 @ 25 ℃ ）， 5 mM Na2EDTA ， 4 ℃ 保存，请勿冻存。 |
| 热失活70 ℃ 10 分钟。 |
| 使用事项已知 PNGase F 的活性受 SDS 的抑制，所以在反应混合物中必须加入 NP-40 。要使天然糖蛋白去糖基化，或许需要增加酶量和延长温育时间。 |
酶解条件
2013 - CC - 邹汉法 - ordered mesoporous silica-carbon NPs 富集糖
The serum samples from 16 healthy volunteers were thawed and centrifuged at 12,000g for 10 min. The collected supernatants (50 μL) were added into ammonium bicarbonate (10 mM, pH 7.5, 450 μL) and denatured in boil water for 5 min. Then ultra-filtration was employed to remove the endogenous peptides by using a membrane (MWCO, 10 KDa) at 14,000 g for 20min.
The collected proteins were washed by ammonium bicarbonate (400 μL) for additional three times, and dissolved in ammonium bicarbonate (10 mM, pH 7.5, 500 μL). Proteins (serum or ovalbumin) were dissolved in 10 mM ammonium bicarbonate solution (pH 7.5) and boiled for 5 min to denature the activity. Then PNGase F (10 U) was added into the mixtures and incubated at 37°C overnight.
The protein digestion (10 μL) was mixed with materials (10 mg/mL, 100 μL) aqueous solution and deionized water (40 μL). After incubated for 20 min, the supernatant by centrifuging at 20,000 g for 3 min were removed, and the precipitation was washed with deionized water (100 μL) for three times. Finally, the glycans were eluted by using 50 % ACN (10 μL) solution for MS analysis.
2011 - AC - 吴仁安; 邹汉法 - 介孔碳 体积排阻 富集N-糖 -7721
Releasing of N-Linked Glycans from Proteins.
Standard glycoproteins with the concentrations of 1 mg/mL were prepared in 10 mM ammonium bicarbonate solution (pH 7.5). The release of the N-linked glycans from the proteins was carried out as follows: the protein solutions (100μL) were boiled for 5 min, cooled to room temperature, and incubated at 37 ºC for 24 h after adding 10 U of PNGase F.
To release the N-linked glycans from human sera, the serum samples from healthy volunteers and liver cancer patients were thawed at 4 ºC and centrifuged at 12 000g for 10 min. The collected supernatants (50 μL) were diluted to 500μL by 10 mM ammonium bicarbonate (pH 7.5), and the samples were ultrafiltrated through a membrane with MWCO of 10 kDa at 14 000g for 20 min. The collected proteins on the membrane were rinsed by with 500 μL of 10mM ammonium bicarbonate (500μL, pH 7.5) for three times and dissolved in 500μL of 10 mM ammonium bicarbonate (10 mM, pH 7.5). The other procedures of releasing glycans from the collected human serum proteins were the same as for the above standard proteins.
Enrichment of N-Linked Glycans.
A10μL of protein digestion was mixed with 10μL of O-CMK-3 (30 mg/mL) aqueous solution and 30μL of deionized water. After being incubated at room temperature for 0.5 h and followed by the removal of supernatant by centrifuging at 20 000g for 2 min, the precipitate was washed with 30 μL of deionized water for three times. Finally, the adsorbed glycans on O-CMK-3 materials were eluted using 10μL of 50% ACN solution
2003 nbt829 Lectin affinity capture, isotope-coded tagging and mass spectrometry to identify N-linked glycoproteins -Hiroyuki Kaji1@Toshiaki Isobe-p
The peptides were then dissolved in 0.1 M Tris base prepared with H218O, and adjusted to pH 8–9 with acetic acid.
PNGase F (lyophilized; Takara) dissolved in H218O was added to the peptide solution (1 mU/10 µg peptide) and the reaction was allowed to proceed overnight at 37 °C in a sealed polypropylene tube.
ac050554q
http://pubs.acs.org/doi/full/10.1021/ac050554q
1-mL aliquot of human serum
~6-8mg Serum total protein
Deglycosylation by PNGase F. Glycopeptides were vacuum-dried and redissolved in 100 μL of 100 mM sodium phosphate buffer (pH 7.5). The reaction mixture was incubated with 2 μL (10 units) of PNGase F for 24 h at 37 °C.
nmeth.3366
The beads were washed with 40 mM ammonium carbonate in 18O-water (1 × 40 μL). N-glycosylated peptides were cleaved by treatment with PNGase F (5 U) in ammonium carbonate in 18O-water (40 mM, 100 μL) in a 10-kDa spin filter (Corning) for 6 h at 37 °C.
