Assessment of TMT Labeling Efficiency in Large-Scale Quantitative Proteomics: The Critical Effect of Sample pH.
Assessment of TMT Labeling Efficiency in Large-Scale Quantitative Proteomics: The Critical Effect of Sample pH.
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DOI：
10.1021/acsomega.1c00776
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发表时间：
2021-05-18
期刊：
影响因子：
4.1
通讯作者：
Adkins JN
中科院分区：
文献类型：
--
作者：
Hutchinson-Bunch C;Sanford JA;Hansen JR;Gritsenko MA;Rodland KD;Piehowski PD;Qian WJ;Adkins JN
通过串联质量标签 (TMT) 试剂进行同量异位标记，可以在 LC-MS/MS 之前对样品进行多重分析，从而促进高通量大规模定量蛋白质组学。一致且高效的标记反应对于实现可靠的定量至关重要；因此，我们的临床蛋白质组学方案中嵌入了质量控制 (QC) 样品，其中包含 TMT 组中每个样品的一小部分，称为“混合 QC”。这种混合 QC 可以在合并完整样品之前通过 LC-MS/MS 检测 TMT 标记问题，从而挽救不良的 TMT 标记反应。虽然 TMT 标记是一种有价值的工具，但导致不良反应的因素尚未得到充分研究。我们观察到，重新标记不一定能挽救 TMT 反应，并且肽样品在 50 mM HEPES 缓冲液（pH 8.5）中重悬后有时仍保持酸性，这与低标记效率 (LE) 和相对较低的中位报告离子强度 (MRII) 相一致。为了获得更具弹性的 TMT 标记程序，我们研究了 LE、报告离子缺失、平均 TMT 组 MRII 与单个通道 MRII 的比率，以及混合 QC 样品的 log2 报告离子比率的分布。我们发现样品 pH 值是 LE 中的关键因素，在重新标记之前增加标记不良的样品中的缓冲液浓度可成功挽救 TMT 标记反应。此外，将肽重悬于 500 mM HEPES 缓冲液中进行 TMT 标记可始终获得较高的 LE 和较低的缺失数据。通过更好地控制标记样品的 pH 值并在合并样品之前实施多种评估标记质量的方法，我们证明了可以实现用于大规模定量研究的稳健 TMT 标记。
Isobaric labeling via tandem mass tag (TMT) reagents enables sample multiplexing prior to LC–MS/MS, facilitating high-throughput large-scale quantitative proteomics. Consistent and efficient labeling reactions are essential to achieve robust quantification; therefore, embedded in our clinical proteomic protocol is a quality control (QC) sample that contains a small aliquot from each sample within a TMT set, referred to as “Mixing QC.” This Mixing QC enables the detection of TMT labeling issues by LC–MS/MS before combining the full samples to allow for salvaging of poor TMT labeling reactions. While TMT labeling is a valuable tool, factors leading to poor reactions are not fully studied. We observed that relabeling does not necessarily rescue TMT reactions and that peptide samples sometimes remained acidic after resuspending in 50 mM HEPES buffer (pH 8.5), which coincided with low labeling efficiency (LE) and relatively low median reporter ion intensities (MRIIs). To obtain a more resilient TMT labeling procedure, we investigated LE, reporter ion missingness, the ratio of mean TMT set MRII to individual channel MRII, and the distribution of log 2 reporter ion ratios of Mixing QC samples. We discovered that sample pH is a critical factor in LE, and increasing the buffer concentration in poorly labeled samples before relabeling resulted in the successful rescue of TMT labeling reactions. Moreover, resuspending peptides in 500 mM HEPES buffer for TMT labeling resulted in consistently higher LE and lower missing data. By better controlling the sample pH for labeling and implementing multiple methods for assessing labeling quality before combining samples, we demonstrate that robust TMT labeling for large-scale quantitative studies is achievable.
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影响因子：
14.8
作者：
Mertins P;Tang LC;Krug K;Clark DJ;Gritsenko MA;Chen L;Clauser KR;Clauss TR;Shah P;Gillette MA;Petyuk VA;Thomas SN;Mani DR;Mundt F;Moore RJ;Hu Y;Zhao R;Schnaubelt M;Keshishian H;Monroe ME;Zhang Z;Udeshi ND;Mani D;Davies SR;Townsend RR;Chan DW;Smith RD;Zhang H;Liu T;Carr SA
通讯作者：
Carr SA
影响因子：
48
作者：
Mertins, Philipp;Qiao, Jana W.;Patel, Jinal;Udeshi, Namrata D.;Clauser, Karl R.;Mani, D. R.;Burgess, Michael W.;Gillette, Michael A.;Jaffe, Jacob D.;Carr, Steven A.
通讯作者：
Carr, Steven A.
影响因子：
64.5
作者：
Zhang H;Liu T;Zhang Z;Payne SH;Zhang B;McDermott JE;Zhou JY;Petyuk VA;Chen L;Ray D;Sun S;Yang F;Chen L;Wang J;Shah P;Cha SW;Aiyetan P;Woo S;Tian Y;Gritsenko MA;Clauss TR;Choi C;Monroe ME;Thomas S;Nie S;Wu C;Moore RJ;Yu KH;Tabb DL;Fenyö D;Bafna V;Wang Y;Rodriguez H;Boja ES;Hiltke T;Rivers RC;Sokoll L;Zhu H;Shih IM;Cope L;Pandey A;Zhang B;Snyder MP;Levine DA;Smith RD;Chan DW;Rodland KD;CPTAC Investigators
通讯作者：
CPTAC Investigators
影响因子：
7.4
作者：
Thompson, A;Schäfer, J;Hamon, C
通讯作者：
Hamon, C
影响因子：
12.3
作者：
Budnik B;Levy E;Harmange G;Slavov N
通讯作者：
Slavov N