Rapid identification of fluorochrome modification sites in proteins by LC ESI-Q-TOF mass spectrometry.
Rapid identification of fluorochrome modification sites in proteins by LC ESI-Q-TOF mass spectrometry.
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DOI：
10.1021/bc100560c
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发表时间：
2011-07-20
影响因子：
4.7
通讯作者：
Siahaan, Teruna J.
中科院分区：
文献类型：
--
作者：
Manikwar, Prakash;Zimmerman, Tahl;Blanco, Francisco J.;Williams, Todd D.;Siahaan, Teruna J.
荧光染料或药物分子与蛋白质赖氨酸残基的ε-氨基的缀合在生物学和医学中具有许多应用。然而，这种类型的缀合产生蛋白质缀合物的异质群体。由于荧光染料或药物分子与蛋白质的结合可能对蛋白质功能产生有害影响，因此有必要鉴定结合位点。不幸的是，鉴定过程可能是耗时且费力的;因此，需要开发快速且可靠的方法来确定荧光标记或药物分子的缀合位点。采用电喷雾四极杆飞行时间质谱（ESI-Q-TOF MS）沿着胰蛋白酶消化肽图分析技术，研究了荧光素-5 ′-异硫氰酸酯和罗丹明-B-异硫氰酸酯与淋巴细胞功能相关抗原-1（LFA-1）α亚基插入结构域（I-domain）蛋白上游离氨基的结合位点。在Q-TOF的碰撞池中生成的荧光染料部分的报告片段（无明确的MS/MS前体选择）用于鉴定缀合位点。报告离子的选定离子图容易标记复合物消化物色谱图中的修饰肽。这些光谱的询问揭示了鉴定修饰肽的中性损失/前体对。结果表明，1至7个荧光素分子或1至4个罗丹明分子连接到I结构域蛋白的赖氨酸残基上。金属离子依赖性粘附位点（MIDAS），这是一个重要的结合区域的I结构域中没有发现修改。
Conjugation of either a fluorescent dye or a drug molecule to the ε-amino groups of lysine residues of proteins has many applications in biology and medicine. However, this type of conjugation produces a heterogeneous population of protein conjugates. Because conjugation of fluorochrome or drug molecule to a protein may have deleterious effects on protein function, the identification of conjugation sites is necessary. Unfortunately, the identification process can be time-consuming and laborious; therefore, there is a need to develop a rapid and reliable way to determine the conjugation sites of the fluorescent label or drug molecule. In this study, the sites of conjugation of fluorescein-5′-isothiocyanate and rhodamine-B-isothiocyanate to free amino groups on the insert-domain (I-domain) protein derived from the α-subunit of lymphocyte function-associated antigen-1 (LFA-1) were determined by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF MS) along with peptide mapping using trypsin digestion. A reporter fragment of the fluorochrome moiety that is generated in the collision cell of the Q-TOF without explicit MS/MS precursor selection was used to identify the conjugation site. Selected ion plots of the reporter ion readily mark modified peptides in chromatograms of the complex digest. Interrogation of theses spectra reveals a neutral loss/precursor pair that identifies the modified peptide. The results show that one to seven fluorescein molecules or one to four rhodamine molecules were attached to the lysine residue(s) of the I-domain protein. No modifications were found in the metal ion-dependent adhesion site (MIDAS), which is an important binding region of the I-domain.
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影响因子：
3
作者：
Wong JW;Schwahn AB;Downard KM
通讯作者：
Downard KM
影响因子：
2
作者：
Bateman, Kevin P.;Castro-Perez, Jose;Nicoll-Griffith, Deborah A.
通讯作者：
Nicoll-Griffith, Deborah A.
影响因子：
4.8
作者：
Stanley, P;Hogg, N
通讯作者：
Hogg, N
DOI：
10.1073/pnas.83.17.6233
发表时间：
1986-09-01
影响因子：
11.1
作者：
HUNT, DF;YATES, JR;HAUER, CR
通讯作者：
HAUER, CR
影响因子：
64.5
作者：
Shimaoka, M;Xiao, T;Springer, TA
通讯作者：
Springer, TA