High-Resolution Enabled TMT 8‑plexing

Isobaric mass tag-based quantitative proteomics strategies such as iTRAQ and TMT utilize reporter ions in the low-mass range of tandem MS spectra for relative quantification. The number of samples that can be compared in a single experiment (multiplexing) is limited by the number of different reporter ions that can be generated by differential stable isotope incorporation (¹⁵N, ¹³C) across the reporter and the mass balancing parts of the reagents. Here, we demonstrate that a higher multiplexing rate can be achieved by utilizing the 6 mDa mass difference between ¹⁵N- and ¹³C-containing reporter fragments, in combination with high-resolution mass spectrometry. Two variants of the TMT127 and TMT129 reagents are available; these are distinguished by the position and the nature of the incorporated stable isotope in the reporter portions of the labels (TMT127L, ¹²C₈H₁₆¹⁵N₁⁺; TMT127H, ¹²C₇¹³C₁H₁₆¹⁴N₁⁺; TMT129L, ¹²C₆¹³C₂H₁₆¹⁵N₁⁺; and TMT129H, ¹²C₅¹³C₃H₁₆¹⁴N₁⁺). We demonstrate that these variants can be baseline-resolved in Orbitrap Elite higher-energy collision-induced dissociation spectra recorded with a 96 ms transient enabling comparable dynamic range, precision, and accuracy of quantification as 1 Da spaced reporter ions. The increased multiplexing rate enabled determination of inhibitor potencies in chemoproteomic kinase assays covering a wider range of compound concentrations in a single experiment, compared to conventional 6-plex TMT-based assays

High-Resolution Enabled TMT 8‑plexing

Isobaric mass tag-based quantitative proteomics strategies such as iTRAQ and TMT utilize reporter ions in the low-mass range of tandem MS spectra for relative quantification. The number of samples that can be compared in a single experiment (multiplexing) is limited by the number of different reporter ions that can be generated by differential stable isotope incorporation (¹⁵N, ¹³C) across the reporter and the mass balancing parts of the reagents. Here, we demonstrate that a higher multiplexing rate can be achieved by utilizing the 6 mDa mass difference between ¹⁵N- and ¹³C-containing reporter fragments, in combination with high-resolution mass spectrometry. Two variants of the TMT127 and TMT129 reagents are available; these are distinguished by the position and the nature of the incorporated stable isotope in the reporter portions of the labels (TMT127L, ¹²C₈H₁₆¹⁵N₁⁺; TMT127H, ¹²C₇¹³C₁H₁₆¹⁴N₁⁺; TMT129L, ¹²C₆¹³C₂H₁₆¹⁵N₁⁺; and TMT129H, ¹²C₅¹³C₃H₁₆¹⁴N₁⁺). We demonstrate that these variants can be baseline-resolved in Orbitrap Elite higher-energy collision-induced dissociation spectra recorded with a 96 ms transient enabling comparable dynamic range, precision, and accuracy of quantification as 1 Da spaced reporter ions. The increased multiplexing rate enabled determination of inhibitor potencies in chemoproteomic kinase assays covering a wider range of compound concentrations in a single experiment, compared to conventional 6-plex TMT-based assays

High-Resolution Enabled TMT 8‑plexing

Isobaric mass tag-based quantitative proteomics strategies such as iTRAQ and TMT utilize reporter ions in the low-mass range of tandem MS spectra for relative quantification. The number of samples that can be compared in a single experiment (multiplexing) is limited by the number of different reporter ions that can be generated by differential stable isotope incorporation (¹⁵N, ¹³C) across the reporter and the mass balancing parts of the reagents. Here, we demonstrate that a higher multiplexing rate can be achieved by utilizing the 6 mDa mass difference between ¹⁵N- and ¹³C-containing reporter fragments, in combination with high-resolution mass spectrometry. Two variants of the TMT127 and TMT129 reagents are available; these are distinguished by the position and the nature of the incorporated stable isotope in the reporter portions of the labels (TMT127L, ¹²C₈H₁₆¹⁵N₁⁺; TMT127H, ¹²C₇¹³C₁H₁₆¹⁴N₁⁺; TMT129L, ¹²C₆¹³C₂H₁₆¹⁵N₁⁺; and TMT129H, ¹²C₅¹³C₃H₁₆¹⁴N₁⁺). We demonstrate that these variants can be baseline-resolved in Orbitrap Elite higher-energy collision-induced dissociation spectra recorded with a 96 ms transient enabling comparable dynamic range, precision, and accuracy of quantification as 1 Da spaced reporter ions. The increased multiplexing rate enabled determination of inhibitor potencies in chemoproteomic kinase assays covering a wider range of compound concentrations in a single experiment, compared to conventional 6-plex TMT-based assays

High-Resolution Enabled TMT 8‑plexing

Isobaric mass tag-based quantitative proteomics strategies such as iTRAQ and TMT utilize reporter ions in the low-mass range of tandem MS spectra for relative quantification. The number of samples that can be compared in a single experiment (multiplexing) is limited by the number of different reporter ions that can be generated by differential stable isotope incorporation (¹⁵N, ¹³C) across the reporter and the mass balancing parts of the reagents. Here, we demonstrate that a higher multiplexing rate can be achieved by utilizing the 6 mDa mass difference between ¹⁵N- and ¹³C-containing reporter fragments, in combination with high-resolution mass spectrometry. Two variants of the TMT127 and TMT129 reagents are available; these are distinguished by the position and the nature of the incorporated stable isotope in the reporter portions of the labels (TMT127L, ¹²C₈H₁₆¹⁵N₁⁺; TMT127H, ¹²C₇¹³C₁H₁₆¹⁴N₁⁺; TMT129L, ¹²C₆¹³C₂H₁₆¹⁵N₁⁺; and TMT129H, ¹²C₅¹³C₃H₁₆¹⁴N₁⁺). We demonstrate that these variants can be baseline-resolved in Orbitrap Elite higher-energy collision-induced dissociation spectra recorded with a 96 ms transient enabling comparable dynamic range, precision, and accuracy of quantification as 1 Da spaced reporter ions. The increased multiplexing rate enabled determination of inhibitor potencies in chemoproteomic kinase assays covering a wider range of compound concentrations in a single experiment, compared to conventional 6-plex TMT-based assays

Ion Coalescence of Neutron Encoded TMT 10-Plex Reporter Ions

Isobaric mass tag-based quantitative proteomics strategies such as iTRAQ and TMT utilize reporter ions in the low mass range of tandem MS spectra for relative quantification. The recent extension of TMT multiplexing to 10 conditions has been enabled by utilizing neutron encoded tags with reporter ion <i>m</i>/<i>z</i> differences of 6 mDa. The baseline resolution of these closely spaced tags is possible due to the high resolving power of current day mass spectrometers. In this work we evaluated the performance of the TMT10 isobaric mass tags on the Q Exactive Orbitrap mass spectrometers for the first time and demonstrated comparable quantification accuracy and precision to what can be achieved on the Orbitrap Elite mass spectrometers. However, we discovered, upon analysis of complex proteomics samples on the Q Exactive Orbitrap mass spectrometers, that the proximate TMT10 reporter ion pairs become prone to coalescence. The fusion of the different reporter ion signals into a single measurable entity has a detrimental effect on peptide and protein quantification. We established that the main reason for coalescence is the commonly accepted maximum ion target for MS2 spectra of 1e6 on the Q Exactive instruments. The coalescence artifact was completely removed by lowering the maximum ion target for MS2 spectra from 1e6 to 2e5 without any losses in identification depth or quantification quality of proteins

High-Resolution Enabled TMT 8‑plexing

Isobaric mass tag-based quantitative proteomics strategies such as iTRAQ and TMT utilize reporter ions in the low-mass range of tandem MS spectra for relative quantification. The number of samples that can be compared in a single experiment (multiplexing) is limited by the number of different reporter ions that can be generated by differential stable isotope incorporation (¹⁵N, ¹³C) across the reporter and the mass balancing parts of the reagents. Here, we demonstrate that a higher multiplexing rate can be achieved by utilizing the 6 mDa mass difference between ¹⁵N- and ¹³C-containing reporter fragments, in combination with high-resolution mass spectrometry. Two variants of the TMT127 and TMT129 reagents are available; these are distinguished by the position and the nature of the incorporated stable isotope in the reporter portions of the labels (TMT127L, ¹²C₈H₁₆¹⁵N₁⁺; TMT127H, ¹²C₇¹³C₁H₁₆¹⁴N₁⁺; TMT129L, ¹²C₆¹³C₂H₁₆¹⁵N₁⁺; and TMT129H, ¹²C₅¹³C₃H₁₆¹⁴N₁⁺). We demonstrate that these variants can be baseline-resolved in Orbitrap Elite higher-energy collision-induced dissociation spectra recorded with a 96 ms transient enabling comparable dynamic range, precision, and accuracy of quantification as 1 Da spaced reporter ions. The increased multiplexing rate enabled determination of inhibitor potencies in chemoproteomic kinase assays covering a wider range of compound concentrations in a single experiment, compared to conventional 6-plex TMT-based assays

High-Resolution Enabled TMT 8‑plexing

Isobaric mass tag-based quantitative proteomics strategies such as iTRAQ and TMT utilize reporter ions in the low-mass range of tandem MS spectra for relative quantification. The number of samples that can be compared in a single experiment (multiplexing) is limited by the number of different reporter ions that can be generated by differential stable isotope incorporation (¹⁵N, ¹³C) across the reporter and the mass balancing parts of the reagents. Here, we demonstrate that a higher multiplexing rate can be achieved by utilizing the 6 mDa mass difference between ¹⁵N- and ¹³C-containing reporter fragments, in combination with high-resolution mass spectrometry. Two variants of the TMT127 and TMT129 reagents are available; these are distinguished by the position and the nature of the incorporated stable isotope in the reporter portions of the labels (TMT127L, ¹²C₈H₁₆¹⁵N₁⁺; TMT127H, ¹²C₇¹³C₁H₁₆¹⁴N₁⁺; TMT129L, ¹²C₆¹³C₂H₁₆¹⁵N₁⁺; and TMT129H, ¹²C₅¹³C₃H₁₆¹⁴N₁⁺). We demonstrate that these variants can be baseline-resolved in Orbitrap Elite higher-energy collision-induced dissociation spectra recorded with a 96 ms transient enabling comparable dynamic range, precision, and accuracy of quantification as 1 Da spaced reporter ions. The increased multiplexing rate enabled determination of inhibitor potencies in chemoproteomic kinase assays covering a wider range of compound concentrations in a single experiment, compared to conventional 6-plex TMT-based assays

Measuring and Managing Ratio Compression for Accurate iTRAQ/TMT Quantification

Isobaric mass tagging (e.g., TMT and iTRAQ) is a precise and sensitive multiplexed peptide/protein quantification technique in mass spectrometry. However, accurate quantification of complex proteomic samples is impaired by cofragmentation of peptides, leading to systematic underestimation of quantitative ratios. Label-free quantification strategies do not suffer from such an accuracy bias but cannot be multiplexed and are less precise. Here, we compared protein quantification results obtained with these methods for a chemoproteomic competition binding experiment and evaluated the utility of measures of spectrum purity in survey spectra for estimating the impact of cofragmentation on measured TMT-ratios. While applying stringent interference filters enables substantially more accurate TMT quantification, this came at the expense of 30%–60% fewer proteins quantified. We devised an algorithm that corrects experimental TMT ratios on the basis of determined peptide interference levels. The quantification accuracy achieved with this correction was comparable to that obtained with stringent spectrum filters but limited the loss in coverage to <10%. The generic applicability of the fold change correction algorithm was further demonstrated by spiking of chemoproteomics samples into excess amounts of <i>E. coli</i> tryptic digests