Pereira-Medrano Ana Gabriela, Sterling Alistair, Snijders Ambrosius P L, Reardon Kenneth F, Wright Phillip C
Biological and Environmental Systems Group, Department of Chemical and Process Engineering, University of Sheffield, Sheffield, United Kingdom.
J Am Soc Mass Spectrom. 2007 Sep;18(9):1714-25. doi: 10.1016/j.jasms.2007.06.011. Epub 2007 Jun 30.
HPLC-MS/MS is widely used for protein identification from gel spots and shotgun fractions. Although HPLC has well recognized benefits, this type of sample infusion also has some undesirable attributes: relatively low sample throughput, potential sample-to-sample carryover, time-varying sample composition, and no option for longer sample infusion for longer MS analyses. An automated chip-based ESI device (CB-ESI) has the potential to overcome these limitations. This report describes a systematic evaluation of the information-dependant acquisition (IDA) and sample preparation protocols for rapid protein identification from a complex mixture using a CB-ESI source compared with HPLC-ESI (gradient and isocratic elutions). Cytochrome c and a six-protein mixture (11-117 kDa) were used to develop an IDA protocol for rapid protein identification and to evaluate the effects of sample preparation protocols. MS (1-10 s) and MS/MS (1-60 s) scan times, sample concentration (50-500 fmol/microL), and ZipTipC(18) cleanup were evaluated. Based on MOWSE scores, protein coverage, experimental run time, number of identified proteins, and reproducibility, a 12.5 min experiment (22 cycles, each with one 3 s MS and eight 10 s MS/MS scans) was determined to be the optimal IDA protocol for CB-ESI. This work flow yielded up to 220% greater peptide coverage compared with gradient HPLC-ESI and provided protein identifications with up to a 2-fold higher throughput rate than either HPLC-ESI approach, whilst employing half the amount of sample over the same time frame. The results from this study support the use of CB-ESI as a rapid alternative to the identification of protein mixtures.
高效液相色谱-串联质谱法（HPLC-MS/MS）被广泛用于从凝胶斑点和鸟枪法馏分中鉴定蛋白质。尽管高效液相色谱具有公认的优点，但这种进样方式也有一些不良特性：相对较低的样品通量、潜在的样品间残留、随时间变化的样品组成，以及无法进行更长时间的进样以进行更长时间的质谱分析。基于芯片的自动电喷雾电离装置（CB-ESI）有可能克服这些限制。本报告描述了一种系统评估，即与HPLC-ESI（梯度洗脱和等度洗脱）相比，使用CB-ESI源从复杂混合物中快速鉴定蛋白质的信息依赖型采集（IDA）和样品制备方案。使用细胞色素c和一种六蛋白混合物（11-117 kDa）来开发一种用于快速蛋白质鉴定的IDA方案，并评估样品制备方案的效果。对质谱（1-10秒）和串联质谱（1-60秒）扫描时间、样品浓度（50-500飞摩尔/微升）以及ZipTipC(18)净化进行了评估。基于MOWSE评分、蛋白质覆盖率、实验运行时间、鉴定出的蛋白质数量和重现性，确定了一个12.5分钟的实验（22个循环，每个循环包括一次3秒的质谱扫描和八次10秒的串联质谱扫描）为CB-ESI的最佳IDA方案。与梯度HPLC-ESI相比，该工作流程产生的肽段覆盖率提高了220%，并且在相同时间内，提供的蛋白质鉴定通量比任何一种HPLC-ESI方法都高出两倍，同时使用的样品量减少了一半。本研究结果支持将CB-ESI作为鉴定蛋白质混合物的一种快速替代方法。
