Moskovets Eugene, Chen Hsuan-Shen, Pashkova Anna, Rejtar Tomas, Andreev Viktor, Karger Barry L
Barnett Institute and Department of Chemistry, Northeastern University, Boston, MA 02115, USA.
Rapid Commun Mass Spectrom. 2003;17(19):2177-87. doi: 10.1002/rcm.1158.
Close deposition of the sample and external standard was used in axial matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to achieve mass accuracy equivalent to that obtained with an internal standard across the entire MALDI plate. In this work, the sample and external standard were deposited by continuous deposition in separate traces, each approximately 200 micro m wide. The dependence of the mass accuracy on the distance between the sample and standard traces was determined across a MALDI target plate with dimensions of 57.5 mm x 57.0 mm by varying the gap between the traces from 100 micro m to 4 mm. During acquisition, two adjacent traces were alternately irradiated with a 200-Hz laser, such that the peaks in the resulting mass spectra combined the sample and external standard. Ion suppression was not observed even when the peptide concentrations in the two traces differed by more than two orders of magnitude. The five peaks from the external standard trace were used in a four-term mass calibration of the masses of the sample trace. The average accuracy across the whole plate with this method was 5 ppm when peaks of the sample trace had signal-to-noise ratios of at least 30 and the gap between the traces was approximately 100 micro m. This approach was applied to determining peptide masses of a reversed-phase liquid chromatographic (LC) separation of a tryptic digest of beta-galactosidase deposited as a long serpentine trace across the MALDI plate, with accuracy comparable to that obtainable using internal calibration. In addition, the eluent from reversed-phase LC separation of a strong cation-exchange fraction containing tryptic peptides from a yeast lysate along with the closely placed external standard was deposited on the MALDI plate. The data obtained in the MS and MS/MS modes on a MALDI-TOF/TOF mass spectrometer were combined and used in database searching with MASCOT. Since the significant score is a function of mass accuracy in the MS mode, database searching with high mass accuracy reduced the number of false positives and also added peptides which otherwise would have been eliminated at lower mass accuracy (false negatives).
在轴向基质辅助激光解吸/电离飞行时间质谱（MALDI-TOFMS）中，采用样品和外标紧密沉积的方式，以在整个MALDI板上实现与内标相当的质量准确度。在本工作中，样品和外标通过连续沉积形成单独的痕迹，每条痕迹宽约200μm。通过将痕迹间的间隙从100μm变化到4mm，在尺寸为57.5mm×57.0mm的MALDI靶板上确定质量准确度对样品和标准痕迹间距离的依赖性。在采集过程中，用200Hz的激光交替照射两条相邻的痕迹，使得所得质谱中的峰结合了样品和外标。即使两条痕迹中的肽浓度相差超过两个数量级，也未观察到离子抑制现象。外标痕迹的五个峰用于对样品痕迹的质量进行四项质量校准。当样品痕迹的峰信噪比至少为30且痕迹间间隙约为100μm时，用此方法在整个板上的平均准确度为5ppm。该方法应用于测定β-半乳糖苷酶胰蛋白酶消化产物的反相液相色谱（LC）分离肽质量，该产物以长蛇形痕迹沉积在MALDI板上，准确度与使用内标校准相当。此外，将来自酵母裂解物的含胰蛋白酶肽的强阳离子交换级分的反相LC分离洗脱液与紧密放置的外标一起沉积在MALDI板上。在MALDI-TOF/TOF质谱仪上以MS和MS/MS模式获得的数据进行合并，并用于用MASCOT进行数据库搜索。由于显著得分是MS模式下质量准确度的函数，高质量准确度的数据库搜索减少了假阳性的数量，还增加了在较低质量准确度下会被排除的肽（假阴性）。
