Optimization for peptide sample preparation for urine peptidomics.
Optimization for peptide sample preparation for urine peptidomics.
复制标题
DOI：
10.1186/1559-0275-11-7
复制
发表时间：
2014-02-25
影响因子：
3.8
通讯作者：
Sarwal MM
中科院分区：
文献类型：
--
作者：
Sigdel TK;Nicora CD;Hsieh SC;Dai H;Qian WJ;Camp DG 2nd;Sarwal MM
分析生物体液中的天然或内源性肽可以为疾病机制提供有价值的见解。此外，检测到的肽也可以作为潜在的生物标志物用于非侵入性监测人类疾病。尿液收集的非侵入性性质和尿液中丰富的肽使得通过高通量“肽组学”方法进行分析成为研究肾脏疾病发病机制的有吸引力的方法。然而，尿肽组学方法在与样品制备相关的困难方面可能存在问题。尿液基质可以在进行分析测量时提供显著的背景干扰，当利用基于LC-MS的肽组分析时，其妨碍肽的鉴定和肽组读数的深度。我们报告了一种新的适应的标准固相萃取（SPE）方法的改进SPE（mSPE）的方法，以提高肽产量和分析灵敏度与LC-MS基于肽组学的时间，成本，堵塞的LC-MS柱，肽产量，肽的质量，并确定每种方法的肽的数量。SPE和mSPE的时间和时间要求相当，但在SPE制备物中明显存在来自尿基质的更多干扰污染物（例如，LC-MS柱堵塞，由于保留的尿胆素导致制备的样品呈淡黄色背景色，肽产率较低）。当我们比较来自4次运行的技术重复的数据时，mSPE方法为从尿液制备样品提供了显著提高的效率（例如，mSPE肽鉴定率为82%，SPE为18%; p = 8.92 E-05）。此外，当应用mSPE方法时，肽鉴定突出了急性肾移植排斥期间尿肽酶的差异活化的生物学，具有特异性肽的明显阶梯，当利用常规SPE方法时，这对于大多数蛋白质是模糊的。总之，当应用LC-MS肽组学分析时，发现mSPE方法上级于用于尿肽样品制备的常规标准SPE方法，这是由于优化的样品净化提供了来自确信鉴定的肽的改进的实验推断。
Analysis of native or endogenous peptides in biofluids can provide valuable insights into disease mechanisms. Furthermore, the detected peptides may also have utility as potential biomarkers for non-invasive monitoring of human diseases. The non-invasive nature of urine collection and the abundance of peptides in the urine makes analysis by high-throughput ‘peptidomics’ methods , an attractive approach for investigating the pathogenesis of renal disease. However, urine peptidomics methodologies can be problematic with regards to difficulties associated with sample preparation. The urine matrix can provide significant background interference in making the analytical measurements that it hampers both the identification of peptides and the depth of the peptidomics read when utilizing LC-MS based peptidome analysis. We report on a novel adaptation of the standard solid phase extraction (SPE) method to a modified SPE (mSPE) approach for improved peptide yield and analysis sensitivity with LC-MS based peptidomics in terms of time, cost, clogging of the LC-MS column, peptide yield, peptide quality, and number of peptides identified by each method. Expense and time requirements were comparable for both SPE and mSPE, but more interfering contaminants from the urine matrix were evident in the SPE preparations (e.g., clogging of the LC-MS columns, yellowish background coloration of prepared samples due to retained urobilin, lower peptide yields) when compared to the mSPE method. When we compared data from technical replicates of 4 runs, the mSPE method provided significantly improved efficiencies for the preparation of samples from urine (e.g., mSPE peptide identification 82% versus 18% with SPE; p = 8.92E-05). Additionally, peptide identifications, when applying the mSPE method, highlighted the biology of differential activation of urine peptidases during acute renal transplant rejection with distinct laddering of specific peptides, which was obscured for most proteins when utilizing the conventional SPE method. In conclusion, the mSPE method was found to be superior to the conventional, standard SPE method for urine peptide sample preparation when applying LC-MS peptidomics analysis due to the optimized sample clean up that provided improved experimental inference from the confidently identified peptides.
登录
查看更多内容
影响因子：
8.8
作者：
Shimwell NJ;Bryan RT;Wei W;James ND;Cheng KK;Zeegers MP;Johnson PJ;Martin A;Ward DG
通讯作者：
Ward DG
影响因子：
13.6
作者：
Ling, Xuefeng B.;Sigdel, Tara K.;Sarwal, Minnie M.
通讯作者：
Sarwal, Minnie M.
影响因子：
2.1
作者：
Sigdel, Tara K.;Lau, Ken;Sarwal, Minnie
通讯作者：
Sarwal, Minnie
影响因子：
3.1
作者：
Machtejevas, Egidijus;Marko-Varga, Gyorgy;Unger, Klaus K.
通讯作者：
Unger, Klaus K.
影响因子：
19.6
作者：
Schaub, S;Wilkins, J;Nickerson, P
通讯作者：
Nickerson, P