Nano-high-performance liquid chromatography in combination with nano-electrospray ionization Fourier transform ion-cyclotron resonance mass spectrometry for proteome analysis
Nano-high-performance liquid chromatography in combination with nano-electrospray ionization Fourier transform ion-cyclotron resonance mass spectrometry for proteome analysis
复制标题
DOI：
10.1002/rcm.1049
复制
发表时间：
2003-01-01
影响因子：
2
通讯作者：
Sinz, A
中科院分区：
文献类型：
--
作者：
Ihling, C;Berger, K;Sinz, A
傅里叶变换离子回旋共振（FTICR）质谱法为生物样品的分析提供了几个优点，包括优异的质量分辨率、超高质量测量精度、高灵敏度和宽质量范围。我们报告了一个纳米HPLC系统耦合到一个FTICR质谱仪配备了nano-electrospray源（nano-HPLC/nano-ESI-FTICRMS）的蛋白质组分析的应用。蛋白质组学中的蛋白质鉴定通常通过准确测定酶促蛋白质酶解产生的肽质量并将其与数据库中理论上消化的蛋白质序列进行比较来进行。采用胰蛋白酶不溶消化牛血清白蛋白，优化实验条件和数据处理。切下人甲状腺组织考马斯亮蓝和银染二维（2D）凝胶上的斑点，用胰蛋白酶进行凝胶内消化，随后通过nano-HPLC/nano-ESI-FTICRMS进行分析。此外，我们分析了来自非洲绿色猴肾的COS-6细胞的膜制备物的1D-凝胶带，作为更复杂的蛋白质混合物的一个例子。使用1 mm反相C-18色谱柱进行样品预浓缩，使用反相C-18毛细管色谱柱进行分离，采用水/乙腈梯度洗脱条件，流速为200 nL/min。常规获得小于3 ppm的质量测量精度。对处理原始数据的不同方法进行了比较，以尽可能高的自动化程度鉴定最大数量的肽。从复杂的混合物中平行鉴定低飞摩尔水平的蛋白质使得纳米HPLC/纳米ESI-FTICRMS成为蛋白质组分析的有吸引力的方法。版权所有（C）2003约翰威利父子有限公司。
Fourier transform ion-cyclotron resonance (FTICR) mass spectrometry offers several advantages for the analysis of biological samples, including excellent mass resolution, ultra-high mass measurement accuracy, high sensitivity, and wide mass range. We report the application of a nano-HPLC system coupled to an FTICR mass spectrometer equipped with nanoelectrospray source (nano-HPLC/nano-ESI-FTICRMS) for proteome analysis. Protein identification in proteomics is usually conducted by accurately determining peptide masses resulting from enzymatic protein digests and comparing them with theoretically digested protein sequences from databases. A tryptic insolution digest of bovine serum albumin was used to optimize experimental conditions and data processing. Spots from Coomassie Blue and silver-stained two-dimensional (2D) gels of human thyroid tissue were excised, in-gel digested with trypsin, and subsequently analyzed by nano-HPLC/nano-ESI-FTICRMS. Additionally, we analyzed 1D-gel bands of membrane preparations of COS-6 cells from African green monkey kidney as an example of more complex protein mixtures. Nano-HPLC was performed using 1-mm reverse-phase C-18 columns for pre-concentration of the samples and reverse-phase C-18 capillary columns for separation, applying water/acetonitrile gradient elution conditions at flow rates of 200 nL/min. Mass measurement accuracies smaller than 3 ppm were routinely obtained. Different methods for processing the raw data were compared in order to identify a maximum number of peptides with the highest possible degree of automation. Parallel identification of proteins from complex mixtures down to low-femtomole levels makes nano-HPLC/nano-ESI-FTICRMS an attractive approach for proteome analysis. Copyright (C) 2003 John Wiley Sons, Ltd.