Precision of Heavy-Light Peptide Ratios Measured by MALDI-TOF Mass Spectrometry
Precision of Heavy-Light Peptide Ratios Measured by MALDI-TOF Mass Spectrometry
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DOI：
10.1021/pr201092v
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发表时间：
2012-03-01
影响因子：
4.4
通讯作者：
Suckau, Detlef
中科院分区：
文献类型：
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作者：
Anderson, N. Leigh;Razavi, Morteza;Suckau, Detlef
我们用6对蛋白质型多肽(LIGH)和同序列稳定同位素标记的合成内标(HIG)考察了MALDI-TOF质谱仪(MS)对多肽定量的精密度。这些被组合成两种类型的稀释曲线，跨越100倍和2000倍的比率。使用反射器采集方法在复制的MALDI斑点上检查变异系数(CV；标准偏差除以平均值)，每个总和光谱中最强的峰需要100000个计数。在100倍稀释曲线的11个点和所有6个多肽上，在每个样本的4个重复点上测定的轻肽/重肽质心峰面积比的变异系数在每个点施加的每个肽的总(轻+重)55fmoL时为2.2%(在多肽中从1.5%到3.7%不等)，在11fmoL时为2.5%。这六个多肽在接近相等(轻=重，稀释曲线中心)下测量的平均变异系数为1.0%，比当五个多肽与第六个多肽(即不同序列的内标)之比时观察到的变异系数低17倍。百倍范围内的响应曲线不是完全线性的，但可以用给出所有多肽的R-2值>；0.998的幂函数拟合来近似模拟。采用MALDI-TOF MS法测定血浆中人蛋白C抑制物(PCI)蛋白型肽(EDQYHYLLDR)的含量。经SISCAPA-MALDI-TOF测定，PCI值为770 ng/m L，重复性测定变异系数为1.5%，靶标浓度为14 000倍。这些结果表明，MALDI-TOF技术可以在100倍的动态范围内提供高到中等丰度的多肽生物标记物的精确定量，当与相同序列标记的内部标准物比例时，并通过特定抗体捕获富集到接近纯度。与传统的纳米LC-MS技术相比，MALDI-TOF的稳健性和吞吐量可以使目前不切实际的大规模蛋白质生物标志物验证研究成为可能。
We have investigated the precision of peptide quantitation by MALDI-TOF mass spectrometry (MS) using six pairs of proteotypic peptides (light) and same-sequence stable isotope labeled synthetic internal standards (heavy). These were combined in two types of dilution curves spanning 100-fold and 2000-fold ratios. Coefficients of variation (CV; standard deviation divided by mean value) were examined across replicate MALDI spots using a reflector acquisition method requiring 100 000 counts for the most intense peak in each summed spectrum. The CV of light/heavy peptide centroid peak area ratios determined on four replicate spots per sample, averaged across 11 points of a 100-fold dilution curve and over all six peptides, was 2.2% (ranging from 1.5 to 3.7% among peptides) at 55 fmol total (light + heavy) of each peptide applied per spot, and 2.5% at 11 fmol applied. The average CV of measurements at near-equivalence (light = heavy, the center of the dilution curve) for the six peptides was 1.0%, about 17-fold lower CV than that observed when five peptides were ratioed to a sixth peptide (i.e., a different-sequence internal standard). Response curves across the 100-fold range were not completely linear but could be closely modeled by a power law fit giving R-2 values >0.998 for all peptides. The MALDI-TOF MS method was used to determine the endogenous level of a proteotypic peptide (EDQYHYLLDR) of human protein C inhibitor (PCI) in a plasma digest after enrichment by capture on a high affinity antipeptide antibody, a technique called stable isotope standards and capture by anti-peptide antibodies (SISCAPA). The level of PCI was determined to be 770 ng/mL with a replicate measurement CV of 1.5% and a >14 000-fold target enrichment via SISCAPA-MALDI-TOF. These results indicate that MALDI-TOF technology can provide precise quantitation of high-to-medium abundance peptide biomarkers over a 100-fold dynamic range when ratioed to same-sequence labeled internal standards and enriched to near purity by specific antibody capture. The robustness and throughput of MALDI-TOF in comparison to conventional nano-LC-MS technology could enable currently impractical large-scale verification studies of protein biomarkers.