Retention Time Prediction for TMT-Labeled Peptides in Proteomic LC-MS Experiments
journal contributionposted on 01.04.2022, 20:39 by Benilde Mizero, Carina Villacrés, Victor Spicer, Rosa Viner, Julian Saba, Bhavinkumar Patel, Sergei Snovida, Penny Jensen, Andreas Huhmer, Oleg V. Krokhin
We present the first detailed study of chromatographic behavior of peptides labeled with tandem mass tags (TMT and TMTpro) in 2D LC for proteomic applications. Carefully designed experimental procedures have permitted generating data sets of over 100,000 nonlabeled and TMT-labeled peptide pairs for the low pH RP in the second separation dimension and data sets of over 10,000 peptide pairs for high-pH RP, HILIC (amide and silica), and SCX separations in the first separation dimension. The average increase in peptide RPLC (0.1% formic acid) retention upon TMT labeling was found to be 3.3% acetonitrile (linear water/acetonitrile gradients), spanning a range of −4 to 10.3%. In addition to the bulk peptide properties such as length, hydrophobicity, and the number of labeled residues, we found several sequence-dependent features mostly associated with differences in N-terminal chemistry. The behavior of TMTpro-labeled peptides was found to be very similar except for a slightly higher hydrophobicity: an average retention shift of 3.7% acetonitrile. The respective versions of the sequence-specific retention calculator (SSRCalc) model have been developed to accommodate both TMT chemistries, showing identical prediction accuracy (R2 ∼ 0.98) for labeled and nonlabeled peptides. Higher retention for TMT-labeled peptides was observed for high-pH RP and HILIC separations, while SCX selectivity remained virtually unchanged.
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tandem mass tagssecond separation dimensionfirst separation dimensionfirst detailed studyspecific retention calculatorretention time predictionbulk peptide propertiesaverage retention shift000 peptide pairslow ph rp2 </ suplabeled peptide pairsslightly higher hydrophobicityfound several sequencehigher retentionph rppeptide rplcr </average increase>< sup000 nonlabeled− 4terminal chemistrysimilar exceptsilica ),scx separationsrespective versionsproteomic lcproteomic applicationspeptides labelednonlabeled peptidesms experimentslinear waterlabeled residueslabeled peptidesformic aciddata sets2d lc