Increased sensitivity of tryptic peptide detection by MALDI-TOF mass spectrometry is achieved by conversion of lysine to homoarginine
Increased sensitivity of tryptic peptide detection by MALDI-TOF mass spectrometry is achieved by conversion of lysine to homoarginine
复制标题
DOI：
10.1006/abio.2000.4834
复制
发表时间：
2000-12-01
影响因子：
2.9
通讯作者：
Becker, GW
中科院分区：
文献类型：
--
作者：
Hale, JE;Butler, JP;Becker, GW
随着对蛋白质组高通量分析需求的增加，通过“质量指纹”（将蛋白质消化的色氨酸肽的质量与数据库中的理论肽相匹配）来鉴定蛋白质的质谱技术，如基质辅助激光解吸电离飞行时间（MALDI-TOF）正在迅速普及。这在一定程度上是由于技术自动化的能力和质谱获取的快速速度。该技术准确性的一个重要因素是在现有的各种搜索算法中识别的色氨酸的数量。获得的亲本蛋白的序列覆盖率越大，鉴定的可信度就越高。高水平序列覆盖率的一个障碍是MAL]DI-TOF质谱法对含精氨酸肽的偏倚。增加对含赖氨酸肽的敏感性可以增加获得的序列覆盖率。为了达到这一结果，我开发了用o -甲基异脲修饰色氨酸肽中赖氨酸的e -胺基团的条件。所利用的条件导致赖氨酸转化为精氨酸，而肽的胺端没有修饰。改良后的MALDI-TOF质谱法检测多肽的灵敏度显著提高。在脱盐和点染到MALDI-TOF板之前，修饰化学可以应用于色氨酸混合物。这种技术对于鉴别赖氨酸/精氨酸比例高的蛋白质特别有用。(C) 2000年学术出版社。
Mass spectrometric techniques for identification of proteins by "mass fingerprinting" (matching the masses of tryptic peptides fi-om a protein digest to the theoretical peptides in a database) such as matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) are rapidly growing in popularity as the demand for high throughput analysis of the proteome increases. This is due, in part, to the ability to automate the technique and the rapid rate with which mass spectra may be acquired. An important factor in the accuracy of the technique is the number of tryptic peptides that are identified in the various searching algorithms that exist. The greater sequence coverage of the parent protein that is obtained, the higher the level of confidence in the identification that is determined. One impediment to high levels of sequence coverage is the bias of MAL]DI-TOF mass spectrometry to arginine-containing peptides. Increasing the sensitivity to lysine-containing peptides should increase the sequence coverage obtained. In order to achieve this result me have developed conditions to modify the E-amine group of lysine in tryptic peptides with O-methylisourea. The conditions utilized result in the conversion of lysine to homoarginine with no modification of the amine terminus of the peptides. The sensitivity of MALDI-TOF mass spectrometry detection of peptides was increased dramatically following modification. The modification chemistry may be applied to tryptic peptide mixtures prior to desalting and spotting onto MALDI-TOF plates. This technique will be particularly useful for identifying proteins with a high lysine/arginine ratio. (C) 2000 Academic Press.