Low-pH Solid-Phase Amino Labeling of Complex Peptide Digests with TMTs Improves Peptide Identification Rates for Multiplexed Global Phosphopeptide Analysis
Low-pH Solid-Phase Amino Labeling of Complex Peptide Digests with TMTs Improves Peptide Identification Rates for Multiplexed Global Phosphopeptide Analysis
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DOI：
10.1021/acs.jproteome.5b00072
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发表时间：
2015-06-01
影响因子：
4.4
通讯作者：
Thompson, Andrew H.
中科院分区：
文献类型：
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作者：
Boehm, Gitte;Prefot, Petra;Thompson, Andrew H.
我们提出了一种新的串联质量标签固相氨基标记（TMT-SPAL）协议，使用可逆固定的肽十八烷基衍生（C18）的固体支持物。这种方法可以减少复杂方案中所需的步骤数量，节省时间并可能减少样品损失。在我们的全球磷酸肽分析工作流程（SysQuant）中，我们可以从方案中减少24小时，同时增加肽鉴定（20%）并减少副反应。固相标记TMT不需要一些修改典型的标记条件，特别是pH值。它已被发现，完全标记相当于标准的碱性pH值溶液相标记的小和大的样品可以实现在C18树脂在微酸性缓冲液条件下。与标准碱性pH溶液相标记相比，C18上的标记行为得到改善。我们分析了我们的样品的组氨酸，丝氨酸，苏氨酸和酪氨酸标记，以确定过度标记的程度，并观察到高于预期的水平（25%的所有肽光谱匹配（PSM））的过度标记在所有这些氨基酸（主要是在酪氨酸和丝氨酸）在我们的标准溶液相标记协议。通过TMT-SPAL方案中使用的低pH条件，所有这些位点的过度标记大大减少（4倍，至所有PSM的7%）。过度标记似乎代表了一种迄今为止被忽视的机制，与无标记方法相比，NHS激活的TMT标记导致肽识别率降低。我们的研究结果还强调了使用标记方法时搜索数据以进行过度标记的重要性。
We present a novel tandem mass tag solid-phase amino labeling (TMT-SPAL) protocol using reversible immobilization of peptides onto octadecyl-derivatized (C18) solid supports. This method can reduce the number of steps required in complex protocols, saving time and potentially reducing sample loss. In our global phosphopeptide profiling workflow (SysQuant), we can cut 24 h from the protocol while increasing peptide identifications (20%) and reducing side reactions. Solid-phase labeling with TMTs does require some modification to typical labeling conditions, particularly pH. It has been found that complete labeling equivalent to standard basic pH solution-phase labeling for small and large samples can be achieved on C18 resins under slightly acidic buffer conditions. Improved labeling behavior on C18 compared to that with standard basic pH solution-phase labeling is demonstrated. We analyzed our samples for histidine, serine, threonine, and tyrosine labeling to determine the degree of overlabeling and observed higher than expected levels (25% of all peptide spectral matches (PSMs)) of overlabeling at all of these amino acids (predominantly at tyrosine and serine) in our standard solution-phase labeling protocol. Overlabeling at all of these sites is greatly reduced (4-fold, to 7% of all PSMs) by the low-pH conditions used in the TMT-SPAL protocol. Overlabeling seems to represent a so-far overlooked mechanism causing reductions in peptide identification rates with NHS-activated TMT labeling compared to that with label-free methods. Our results also highlight the importance of searching data for overlabeling when labeling methods are used.