In-gel digestion. Gel pieces were i

凝胶内消化。将凝胶碎片在50mM NH4HCO3的50％<br>乙腈（体积/体积）溶液中孵育直至澄清。将凝胶碎片用100μl的100％<br>乙腈脱水5min；然后除去液体，并将凝胶碎片<br>在10mM二硫苏糖醇中再水化，并在56℃下温育60分钟。<br>再次将凝胶碎片在100％乙腈中脱水；然后除去液体，并<br>用55mM碘乙酰胺将凝胶碎片再水化。将样品<br>在黑暗中于25°C 孵育45分钟。凝胶碎片用50mM NH4HCO3洗涤，<br>用100％乙腈脱水，再用10ngμl-1 <br>胰蛋白酶<br>再水化，再悬浮在冰上的50mM NH4HCO3中1h。除去多余的液体，然后<br>凝胶碎片在37°C下用胰蛋白酶消化过夜。肽先后<br>用50％乙腈/ 5％甲酸和100％乙腈提取。将肽<br>干燥至完全，并重悬于2％乙腈/0.1％甲酸中。

凝胶内消化。凝胶片在 50mm Nh4HCO3 中孵育， 在 50%<br>醋酸（卷/卷），直到清楚。凝胶片脱水，100μl的100%<br>醋酸 5 分钟;液体然后被移除，凝胶片<br>在 10mM 二西奥斯赖托中补水，并在 56 °C 下孵育 60 分钟。凝胶片<br>再次脱水在100%乙酰三叶酸;液体然后被移除，<br>凝胶片用 55mM 碘乙酰胺重新水化。样品孵育<br>在黑暗中 25°C 下 45 分钟。凝胶片用 50mM NH4HCO3 洗净，<br>用 100% 乙酰酸化脱水，用 10ng μl=1 重新脱水<br> 胰蛋白酶<br>在 50mm Nh4hco3 冰上重新暂停 1 小时。多余的液体被移除，<br>凝胶片在37°C下被消化。提取肽<br>50% 乙酰甲酸/5% 甲酸，其次是 100% 乙酰三叶酸。肽是<br>干到完成，并在2%乙酰三叶酸/0.1%的甲酸中重新消耗。

In-gel digestion. Gel pieces were incubated in 50mM NH4HCO3 in 50% acetonitrile (vol/vol) until clear. Gel pieces were dehydrated with 100μl of 100% acetonitrile for 5min; the liquid was then removed and the gel pieces were rehydrated in 10mM dithiothreitol and incubated at 56 °C for 60min. Gel pieces were dehydrated again in 100% acetonitrile; the liquid was then removed and gel pieces were rehydrated with 55mM iodoacetamide. Samples were incubated at 25°C in the dark for 45min. Gel pieces were washed with 50mM NH4HCO3, dehydrated with 100% acetonitrile and rehydrated with 10ng μl−1 trypsin resuspended in 50mM NH4HCO3 on ice for 1h. Excess liquid was removed, and gel pieces were digested with trypsin at 37 °C overnight. Peptides were extracted with 50% acetonitrile/5% formic acid, followed by 100% acetonitrile. Peptides were dried to completion and resuspended in 2% acetonitrile/0.1% formic acid.<br>