Lei Zhentian, Anand Ajith, Mysore Kirankumar S, Sumner Lloyd W
Samuel Roberts Noble Foundation, Ardmore, Oklahoma, USA.
Methods Mol Biol. 2007;355:353-63. doi: 10.1385/1-59745-227-0:353.
A method has been developed for the extraction of intact proteins from SDS-PAGE gels and for subsequent MALDI-TOF MS analysis to determine precise molecular mass. The method consists of an electroelution step and a subsequent low-temperature matrix-analyte cocrystallization step prior to MALDI-TOF MS. Proteins were first extracted from polyacrylamide gels using electroelution (ProteoPLUS). Following electroelution, salts, SDS, and dye were removed by dialysis using the same electroelution tubes. The proteins were then concentrated in a vacuum centrifuge. Using bovine serum albumin (BSA) and phosphorylase B (Phos B) as standards, it was found that electroelution was more effective than passive elution in extracting intact proteins from gels. Optimized protein recoveries were determined to be 89% for BSA and 58% for Phos B, supporting the hypothesis of an inverse relationship between recovery and protein size. The traditional practice of "fixing gels" using methanol and acetic acid was found to reduce protein recoveries significantly and should be avoided if possible. A subsequent sample preparation method involving the low-temperature cocrystallization of whole proteins with MALDI matrices (trans-3,5-dimethoxy-4-hydroxycinnamic acid, sinapinic acid, 10 mg/mL) was also developed to remove residual impurities further. Proteins and MALDI matrices were mixed, sealed, and stored at 4 degrees C to allow for overnight cocrystallization of matrix and protein. The supernatant was removed and protein-matrix crystals redissolved using the same matrix solution. MALDI-TOF MS signal intensities were found to increase three- to fourfold using the low-temperature cocrystallization method relative to the more common method of immediate analysis following matrix-analyte mixing and "dried droplet" deposition. The reported method is illustrated using two beta-1,3-endoglucanase isoforms from wheat apoplast extracts.
已开发出一种从SDS - PAGE凝胶中提取完整蛋白质并随后进行MALDI - TOF MS分析以确定精确分子量的方法。该方法包括一个电洗脱步骤以及在MALDI - TOF MS之前的后续低温基质 - 分析物共结晶步骤。首先使用电洗脱（ProteoPLUS）从聚丙烯酰胺凝胶中提取蛋白质。电洗脱后，使用相同的电洗脱管通过透析去除盐、SDS和染料。然后在真空离心机中浓缩蛋白质。以牛血清白蛋白（BSA）和磷酸化酶B（Phos B）作为标准品，发现电洗脱在从凝胶中提取完整蛋白质方面比被动洗脱更有效。确定优化后的蛋白质回收率对于BSA为89%，对于Phos B为58%，支持了回收率与蛋白质大小呈反比关系的假设。发现使用甲醇和乙酸“固定凝胶”的传统做法会显著降低蛋白质回收率，应尽可能避免。还开发了一种后续样品制备方法，涉及完整蛋白质与MALDI基质（反式 - 3,5 - 二甲氧基 - 4 - 羟基肉桂酸、芥子酸，10 mg/mL）的低温共结晶，以进一步去除残留杂质。将蛋白质和MALDI基质混合、密封并在4℃下储存，以使基质和蛋白质过夜共结晶。去除上清液，并使用相同的基质溶液重新溶解蛋白质 - 基质晶体。相对于基质 - 分析物混合和“干滴”沉积后立即分析的更常见方法，发现使用低温共结晶方法MALDI - TOF MS信号强度增加了三到四倍。使用来自小麦质外体提取物的两种β - 1,3 - 内切葡聚糖酶同工型对所报道的方法进行了说明。
