RATIONALE: Isobaric tagging reagents, such as tandem mass tags (TMT) and isobaric tags for relative and absolute quantitation (iTRAQ), are high-throughput methods that allow the analysis of multiple samples simultaneously, which reduces instrument time and error. Accuracy and precision of isobaric tags are limited, however, in tandem mass spectrometry (MS/MS) acquisition due to co-isolation and co-fragmentation of neighboring peptide peaks in precursor scans. Here we present a MS(3) method using pulsed-Q dissociation (PQD) in ion trap and Orbitrap instrumentation as a means to improve ratio distortion and maintain high numbers of identified and quantified proteins. METHODS: Mouse brain protein digests were labeled with TMT-128, 129, 130, 131 reagents, mixed in the following molar ratios 1:1:2:5, respectively, and analyzed using HCD-MS(3) and PQD-MS(3) methods. The most intense fragment ion (termed as HCD-MS(3)-top ion or PQD-MS(3)-top ion) or y1 ion (i.e., lysine-TMT tag ion; termed as HCD-MS(3)-y1 or PQD-MS(3)-y1) in collision-induced dissociation (CID) MS/MS was selected for MS(3). RESULTS: Calculated protein ratios obtained in HCD-MS(3)-top ion and PQD-MS(3)-top ion, HCD-MS(3)-y1, and PQD-MS(3)-y1 are accurate and PQD-MS(3) methods resulted in higher numbers of identified and quantified peptide spectral counts and proteins. CONCLUSIONS: PQD-MS(3) methods increase the amount of MS/MS spectra collected and number of quantified proteins and are accessible to those researchers with not only an orbitrap but also an ion trap mass spectrometer.
RATIONALE: Isobaric tagging reagents, such as tandem mass tags (TMT) and isobaric tags for relative and absolute quantitation (iTRAQ), are high-throughput methods that allow the analysis of multiple samples simultaneously, which reduces instrument time and error. Accuracy and precision of isobaric tags are limited, however, in tandem mass spectrometry (MS/MS) acquisition due to co-isolation and co-fragmentation of neighboring peptide peaks in precursor scans. Here we present a MS(3) method using pulsed-Q dissociation (PQD) in ion trap and Orbitrap instrumentation as a means to improve ratio distortion and maintain high numbers of identified and quantified proteins. METHODS:Mouse brain protein digests were labeled with TMT-128, 129, 130, 131 reagents, mixed in the following molar ratios 1:1:2:5, respectively, and analyzed using HCD-MS(3) and PQD-MS(3) methods. The most intense fragment ion (termed as HCD-MS(3)-top ion or PQD-MS(3)-top ion) or y1 ion (i.e., lysine-TMT tag ion; termed as HCD-MS(3)-y1 or PQD-MS(3)-y1) in collision-induced dissociation (CID) MS/MS was selected for MS(3). RESULTS: Calculated protein ratios obtained in HCD-MS(3)-top ion and PQD-MS(3)-top ion, HCD-MS(3)-y1, and PQD-MS(3)-y1 are accurate and PQD-MS(3) methods resulted in higher numbers of identified and quantified peptide spectral counts and proteins. CONCLUSIONS:PQD-MS(3) methods increase the amount of MS/MS spectra collected and number of quantified proteins and are accessible to those researchers with not only an orbitrap but also an ion trap mass spectrometer.