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The design and synthesis of a proline-based reporter isobaric Tandem Mass Tag structure (TMTpro) is presented. An analysis is made of the performance of the new TMTpro tags in comparison with the current commercially available dimethylpiperidine-reporter-based TMT10/11 reagents. The new reporter structure provides a set of 16 tags for use with resolution of 6.3 mDa mass differences in high resolution mass spectrometers and a set of 9 reagents with 1 Da spacing between reporter ions for single dalton analysis using 9 heavy nuclei per tag. We show similar performance in terms of peptide identification rates and quantification between the TMTpro 16-plex and TMT10/11-plex reagents. We also demonstrate the suitability of the TMTpro reagents for phosphopeptide analysis. The ability to pool 16 samples reduces the overall amount of sample required for each channel, and we anticipate that TMTpro reagents will be a useful enhancement for any protocol that benefits from sample pooling and should reduce missing data.

Isobaric labeling reagents such as Tandem Mass Tags (TMT®) enable the genome-wide quantification of protein expression levels under different conditions using a gel-free MS/MS-based approach. Here, we applied a TMTduplex approach with two isobaric tags to study the response of the human pathogen Neisseria meningitidis to deprivation of iron, a condition met in the human body. In total, 609 proteins were identified in samples of three independent growth experiments, in which we compared cultures grown in the presence and absence of iron. Expression of 35 proteins was found to be induced or repressed under iron-limiting conditions, including 11 proteins whose ORFs were not previously identified in DNA array studies as being regulated by iron availability at the transcriptional level. These 11 proteins include proteins likely involved in iron metabolism. ©2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.