基于荧光的肽标记和分级策略，用于分析含半胱氨酸的肽。,Analytical Chemistry

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基于荧光的肽标记和分级策略，用于分析含半胱氨酸的肽。
Analytical Chemistry ( IF 6.7 ) Pub Date : 2005 Jul 15 , DOI: 10.1021/ac050247k
Adrienne Clements ₁ , Murray V. Johnston ₁ , Barbara S. Larsen ₁ , Charles N. McEwen ₁

Affiliation

这项研究表明1,5-I-AEDANS（5-（{{2-[（（碘代乙酰基氨基）乙基}氨基}萘）萘-1-磺酸）可以用作通用的基于荧光的肽定量工具，并可以轻松提供从头测序可解释的串联质谱。评估了两种AEDANS-半胱氨酰肽分级分离策略。一种AEDANS-半胱氨酰肽分级分离策略采用固定化的金属亲和色谱（IMAC）来回收AEDANS标记的肽并降低肽混合物的复杂性。在另一种固相方法中，将1,5-I-AEDANS与基于邻硝基苄基的光裂解树脂偶联，制得一种树脂，该树脂可以用修饰的AEDANS标记物标记和分离含巯基和半胱氨酸的肽（mAEDANS： 5-（（4-氨基-4-氧代丁酰基）{2-[（碘乙酰基）氨基]乙基}氨基）萘-1-磺酸。该分级方案比IMAC策略更具体地富集了含半胱氨酸的肽。使用带有在线荧光检测器和Q-TOF质谱仪的micro-LC-ESI-MS，我们生成了基于荧光的洗脱曲线和AEDANS标记的肽的相应正离子质谱图。这项研究表明AEDANS肽可产生正离子ESI-MS质谱，其检出限可与未标记肽相比。荧光诱导的AEDANS肽的碰撞诱导解离（CID）显示了带有完整标签的易于解释的产物离子光谱。类似于AEDANS标记的肽，mAEDANS标记的硫醇是荧光的，mAEDANS标记的肽的CID也显示了完整的可解释的产物离子光谱。

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Fluorescence-based peptide labeling and fractionation strategies for analysis of cysteine-containing peptides.

This study demonstrates that 1,5-I-AEDANS (5-({2-[(iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid) can be used as a versatile fluorescence-based peptide quantification tool and provides readily interpretable tandem mass spectra for de novo peptide sequencing. Two AEDANS-cysteinyl-peptide fractionation strategies were evaluated. One AEDANS-cysteinyl-peptide fractionation strategy employs immobilized metal affinity chromatography (IMAC) to recover AEDANS-labeled peptides and reduce the complexity of peptide mixtures. In an alternate solid-phase approach, 1,5-I-AEDANS was coupled to an o-nitrobenzyl-based photocleavable resin to produce a resin that can label and isolate thiols and cysteine-containing peptides with a modified-AEDANS label (mAEDANS: 5-((4-amino-4-oxobutanoyl){2-[(iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfoni c acid). This fractionation protocol enriches cysteine-containing peptides more specifically than the IMAC strategy. Using micro-LC-ESI-MS with an on-line fluorescence detector and a Q-TOF mass spectrometer, we generated fluorescence-based elution profiles and corresponding positive ion mass spectra of AEDANS-labeled peptides. This study demonstrates that AEDANS-peptides produce positive ion ESI-MS mass spectra with detection limits comparable to those of the unlabeled peptide. Collision-induced dissociation (CID) of fluorescent AEDANS-peptides revealed readily interpretable product ion spectra with the label intact. Similar to the AEDANS-labeled peptide, an mAEDANS-labeled thiol is fluorescent and CID of a mAEDANS-labeled peptide also reveals an interpretable product ion spectrum with the label intact.

更新日期：2017-01-31

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