Delmotte Nathanaël, Lasaosa Maria, Tholey Andreas, Heinzle Elmar, van Dorsselaer Alain, Huber Christian G
Department of Chemistry, Instrumental Analysis and Bioanalysis, Saarland University, Saarbrücken, Germany.
J Sep Sci. 2009 Apr;32(8):1156-64. doi: 10.1002/jssc.200800615.
The repeatability of peptide identifications in shotgun proteome analyses employing strong cation-exchange-xion-pair RP HPLC hyphenated to ESI MS/MS was compared to an alternative scheme, comprising high-pH RP chromatography combined with low-pH ion-pair RP chromatography. Equivalent results were obtained with both methods in proteome analysis of Corynebacterium glutamicum. From a total number of 1350 to 1850 peptides identified in triplicate analyses of five consecutive fractions chosen from the first-dimension separation, 41-45% of the peptides were identified three times, whereas 16-22 and 37-39% of the peptides were identified only twice or once, respectively. A comparison of the repeatability of peptide identifications from complex samples upon 1- or 2-D chromatographic separation revealed that an additional separation dimension decreases the repeatability by approximately 25%.
在采用强阳离子交换-离子对反相高效液相色谱联用电喷雾串联质谱的鸟枪法蛋白质组分析中，将肽段鉴定的重复性与另一种方案进行了比较，该方案包括高pH值反相色谱与低pH值离子对反相色谱。在谷氨酸棒杆菌的蛋白质组分析中，两种方法得到了等效结果。在从一维分离中选取的五个连续馏分的三次重复分析中，共鉴定出1350至1850个肽段，其中41%-45%的肽段被鉴定出三次，而分别只有16%-22%和37%-39%的肽段被鉴定出两次或一次。对复杂样品经一维或二维色谱分离后肽段鉴定重复性的比较表明，增加一个分离维度会使重复性降低约25%。
