HCD-only fragmentation method balances peptide identification and quantitation of TMT-labeled samples in hybrid...

Protein quantitation based on the generation of reporter ions from chemical labels is a widely used quantitative proteomics approach that enables measuring changes in protein abundance in response to biological perturbations. Isobaric labeling strategies at the MS2 level allow simultaneous measurements of different samples but it requires a fine-tuning of the collision energy used in HCD fragmentation to simultaneously obtain confident peptide identifications and highly sensitive and accurate quantitation. Although the recent development of dual CID/HCD fragmentation methods to circumvent these limitations, the fact is that many laboratories still use HCD-only methods for routine TMT protein quantitation experiments. Here, we have explored the effect of the collision energy on peptide identification and quantitation using HCD-only fragmentation methods on a linear ion trap/orbitrap mass spectrometer bearing an axial field HCD fragmentation cell. Our results using the HCD-only method show that a balance between the increase in the number of peptide identifications and the decrease in the precision of peptide quantitation is attained at a normalized collision energy of 40%. The HCD-only method at 40% does not only yield better results than those obtained using a higher collision energies, but it also outperforms the results obtained using the available CID/HCD dual method.

In this work we have explored the effect of the collision energy on peptide identification and quantitation using HCD-only fragmentation methods on an Orbitrap Velos Pro mass spectrometer. Our results show that when using a HCD-only method, a balance between the number of peptide identifications and the precision of peptide quantitation is attained at a normalized collision energy (NCE) of 40%. This contrast with the parameters routinely used in many laboratories, which are set at NCE 45%. The single HCD method at 40% does not only yield better results than those obtained using a collision energy of 45% but it also outperforms the results obtained using the available CID/HCD dual method. Therefore, we suggest that the single HCD method using the optimal NCE of 40% can therefore become the method of choice in routinely TMT protein quantitation experiments.