Chalmers Michael J, Håkansson Kristina, Johnson Robert, Smith Richard, Shen Jianwei, Emmett Mark R, Marshall Alan G
National High Magnetic Field Laboratory, Florida State University, Tallahassee, FL 32310-4005, USA.
Proteomics. 2004 Apr;4(4):970-81. doi: 10.1002/pmic.200300650.
A microelectrospray ionization tandem Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS(n)) approach for structural characterization of protein phosphorylation is described. Identification of proteolytic peptides is based solely upon mass measurement by high field (9.4 Tesla) FT-ICR MS. The location of the modification within any phosphopeptide is then established by FT-ICR MS(2) and MS(3) experiments. Structural information is maximized by use of electron capture dissociation (ECD) and/or infrared multiphoton dissociation (IRMPD). The analytical utility of the method is demonstrated by characterization of protein kinase A (PKA) phosphorylation. In a single FT-ICR MS experiment, 30 PKA tryptic peptides (including three phosphopeptides) were mass measured by internal calibration to within an absolute mean error of |0.7 ppm|. The location of each of the three sites of phosphorylation was then determined by MS(2) and MS(3) experiments, in which ECD and IRMPD provide complementary peptide sequence information. In two out of three cases, electron irradiation of a phosphopeptide M + nH ion produced an abundant charge-reduced M + nH ion, but few sequence-specific c and z(*) fragment ions. Subsequent IRMPD (MS(3)) of the charge-reduced radical ion resulted in the detection of a large number of ECD-type ion products (c and z ions), but no b or y type ions. The utility of activated ion ECD for the characterization of tryptic phosphopeptides was then demonstrated.
描述了一种用于蛋白质磷酸化结构表征的微电喷雾电离串联傅里叶变换离子回旋共振质谱法（ESI FT-ICR MS(n)）。蛋白水解肽的鉴定仅基于高场（9.4特斯拉）FT-ICR MS的质量测量。然后通过FT-ICR MS(2)和MS(3)实验确定任何磷酸肽内修饰的位置。通过使用电子捕获解离（ECD）和/或红外多光子解离（IRMPD）使结构信息最大化。通过对蛋白激酶A（PKA）磷酸化的表征证明了该方法的分析效用。在单次FT-ICR MS实验中，通过内部校准对30个PKA胰蛋白酶肽（包括三个磷酸肽）进行质量测量，绝对平均误差在|0.7 ppm|以内。然后通过MS(2)和MS(3)实验确定三个磷酸化位点中每个位点的位置，其中ECD和IRMPD提供互补的肽序列信息。在三分之二的情况下，磷酸肽M + nH离子的电子辐照产生了大量电荷减少的[M + nH]((n - 1)+)离子，但很少有序列特异性的c和z()碎片离子。随后对电荷减少的自由基离子进行IRMPD（MS(3)），检测到大量ECD型离子产物（c和z离子），但没有b或y型离子。然后证明了活化离子ECD用于胰蛋白酶磷酸肽表征的效用。
