Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa , 1955 East-West Road, Honolulu, Hawaii 96822, United States.
Anal Chem. 2013 Sep 17;85(18):8873-80. doi: 10.1021/ac402246r. Epub 2013 Sep 5.
Derivatizations that enhance mass spectral quality often require desalting, which presents as a bottleneck in matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS)-proteomics. Guanidination, which converts lysine to homoarginine, an arginine analogue, can increase detection of those peptides 5-15-fold. Our aim was to improve guanidination by using a novel reagent, O-methylisourea-freebase. In a simple reaction, interfering salts were removed prior to guanidination. Freebase preparation took about 30 min and could be applied to samples all at once as opposed to desalting samples one-by-one for 5 min each. For freebase guanidinated BSA tryptic peptides, more than 6-times the peptides were observed relative to tryptic peptides or those guanidinated with the conventional reagent, O-methylisourea hemisulfate. Peptide signals increased more than 10-fold relative to those from guanidination with the conventional reagent and were equivalent to those from conventional guanidination with desalting. In addition, freebase guanidination allowed for a lower limit of detection when combined with another derivatization, N-terminal sulfonation, as evidenced by tandem mass spectrometry (MS/MS) fragmentation analysis of in-gel digests of cytochrome c. Freebase guanidination of rat lung proteins after 2-D gel electrophoresis allowed for identification of all tested protein spots regardless of protein characteristics (MW or pI) or abundance. Co-derivatization with N-terminal sulfonation confirmed the identity of low-abundance proteins in 2-D gel spots that contained more than one protein. The freebase guanidination reagent is simple to prepare and to implement. Desalting is not needed prior to MALDI-TOF MS. Freebase guanidination effectively increases the dynamic range of detection of lysine-containing peptides while decreasing the work needed for sample preparation.
衍生化可以增强质谱质量，通常需要脱盐，但这在基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）-蛋白质组学中成为一个瓶颈。胍基化可以将赖氨酸转化为同精氨酸，即精氨酸类似物，从而将这些肽的检测量提高 5-15 倍。我们的目的是通过使用一种新的试剂，即无甲氧基异脲游离碱，来改进胍基化。在一个简单的反应中，在胍基化之前去除干扰盐。游离碱的制备大约需要 30 分钟，并且可以一次应用于所有样品，而不是对每个样品进行 5 分钟的脱盐处理。对于游离碱胍基化的 BSA 胰蛋白酶肽，与胰蛋白酶肽或用常规试剂 O-甲基异脲半硫酸盐胍基化的肽相比，观察到的肽超过 6 倍。与用常规试剂胍基化相比，肽信号增加了 10 多倍，与用常规胍基化和脱盐处理的肽信号相当。此外，与另一种衍生化反应，N-端磺化结合使用时，游离碱胍基化可以降低检测下限，这可以通过细胞色素 c 胶内酶解产物的串联质谱（MS/MS）碎裂分析得到证明。大鼠肺蛋白经 2-D 凝胶电泳后用游离碱胍基化，无论蛋白质特性（MW 或 pI）或丰度如何，均可鉴定所有测试的蛋白质斑点。与 N-端磺化的共衍生化证实了含有多种蛋白质的 2-D 凝胶斑点中低丰度蛋白质的身份。游离碱胍基化试剂制备简单，易于实施。在进行 MALDI-TOF MS 之前不需要脱盐。游离碱胍基化有效地增加了含赖氨酸肽的检测动态范围，同时减少了样品制备所需的工作量。
