基于保留时间和质荷比匹配的液相色谱-质谱联用技术用于非标记肽段的差异分析

建立了一种无需化学标记的,基于纳升级毛细管液相色谱-电喷雾离子阱质谱联用技术和质谱数据处理的肽段差异分析方法。本方法采用定量差异分析与肽序列鉴定分析分别进行的策略,首先对样品进行质谱全扫描的液质全谱式分析,在全扫描质谱数据中提取肽特征点信息,通过保留时间和质荷比参数匹配不同样品中的共有肽特征点,比较其相对峰强度有无差异。最后对样品中存在丰度差异的肽特征点进行选择性二级质谱分析和序列鉴定,从而实现复杂样品中肽段的差异比较分析。以血浆蛋白酶解混合物为实验对象,考察了本方法用于肽段相对定量分析的重现性以及浓度信号响应曲线等。结果表明:提取的肽特征点峰强度相对标准偏差的中值<22%,肽段离子强度动态范围达3个数量级,在5～1000fmol范围内对肽段定量具有良好线性关系。本方法可用于不同条件样品中具有倍数差异的内源性肽的比较分析。

Retention Time Mass-charge Ratio Pairs for Label-free Differential Analysis of Peptides

A simple LC-MS based methodology is presented that allows relative changes in abundance of peptides in highly complex mixtures to be determined.Proposed method involves signal extraction,generation of features,alignment of corresponding features across different replicates/samples,and monitoring fold changes in relative abundance among different conditions without requiring the use of isotopic or metabolic labeling strategies.Utilizing a nano-flow chromatographic separation system along with the high mass resolution and mass accuracy of an electrospray-ionization(ESI) ion-trap mass spectrometer,the quantitative comparison of thousands of ions emanating from identically prepared control and experimental samples can be made.Using this configuration,the change in relative abundance of a small number of ions between the two conditions solely can be determined by retention time and m/z value.Employing standard operating procedures for both sample preparation and ESI-mass spectrometry,one typically obtains under 1.34 min retention time precision,0.3 amu mass precision and a median of RSD less than 22%,as well as a good linearity of the response curve for peptide ions over 3 orders of magnitude in concentration.Highly selective identification of discriminatory peptides within a matrix of constitutively represented peptides using targeted MS/MS is achieved.The application of this method may represent a promising approach for quantitative peptidomics and discovery of peptide biomarkers.