Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, Scotland, UK.
Proteomics. 2013 Oct;13(20):2967-75. doi: 10.1002/pmic.201300100.
Proteomic profiling by MALDI-TOF MS presents various advantages (speed of analysis, ease of use, relatively low cost, sensitivity, tolerance against detergents and contaminants, and possibility of automation) and is being currently used in many applications (e.g. peptide/protein identification and quantification, biomarker discovery, and imaging MS). Earlier studies by many groups indicated that moderate reproducibility in relative peptide quantification is a major limitation of MALDI-TOF MS. In the present work, we examined and demonstrate a clear effect, in cases apparently random, of sample dilution in complex samples (urine) on the relative quantification of peptides by MALDI-TOF MS. Results indicate that in urine relative abundance of peptides cannot be assessed with confidence based on a single MALDI-TOF MS spectrum. To account for this issue, we developed and propose a novel method of determining the relative abundance of peptides, taking into account that peptides have individual linear quantification ranges in relation to sample dilution. We developed an algorithm that calculates the range of dilutions at which each peptide responds in a linear manner and normalizes the received peptide intensity values accordingly. This concept was successfully applied to a set of urine samples from patients diagnosed with diabetes presenting normoalbuminuria (controls) and macroalbuminuria (cases).
基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）的蛋白质组学分析具有多种优势（分析速度快、使用方便、成本相对较低、灵敏度高、耐受去污剂和污染物、具有自动化的可能性），目前已广泛应用于多个领域（例如肽/蛋白质的鉴定和定量、生物标志物的发现和成像 MS）。许多研究小组的早期研究表明，MALDI-TOF MS 在相对肽定量方面的中等重现性是其主要限制。在本工作中，我们在复杂样品（尿液）中，检查并证明了在明显随机的情况下，样品稀释对 MALDI-TOF MS 相对定量的明显影响。结果表明，在尿液中，不能仅基于单个 MALDI-TOF MS 谱来确定肽的相对丰度。为了解决这个问题，我们开发并提出了一种新的方法来确定肽的相对丰度，考虑到肽与样品稀释相关，具有个体线性定量范围。我们开发了一种算法，计算每个肽以线性方式响应的稀释范围，并相应地对接收的肽强度值进行归一化。该概念已成功应用于一组来自被诊断为糖尿病且具有正常白蛋白尿（对照）和大量白蛋白尿（病例）的患者的尿液样本。
