Collision energies on QTof and Orbitrap instruments: How to make proteomics measurements comparable?

Abstract

Quadrupole time‐of‐flight (QTof) collision‐induced dissociation (CID) and Orbitrap higher‐energy collisional dissociation (HCD) are the most commonly used fragmentation techniques in mass spectrometry‐based proteomics workflows. The information content of the MS/MS spectra is first and foremost determined by the applied collision energy. How can we set up the two instrument types to achieve maximum transferability? To answer this question, we compared MS/MS spectra obtained on a Bruker QTof CID and a Thermo Q‐Exactive Focus Orbitrap HCD instrument as a function of collision energy using the similarity index. Results show that with a few eV lower collision energy setting on HCD (Orbitrap‐specific CID) than on QTof CID, nearly identical MS/MS spectra can be obtained for leucine enkephalin pentapeptide standard, for selected +2 and +3 enolase tryptic peptides and for a large number of peptides in a HeLa protein digest. The Bruker QTof was able to produce colder ions, which may be significant to study inherently labile compounds. Further, we examined energy dependence of peptide identification confidence, as characterized by Mascot scores, on the HeLa peptides. In line with earlier QTof results, this dependence shows one or two maxima (unimodal or bimodal behavior) on Orbitrap. The fraction of bimodal peptides is lower on Orbitrap. Optimal energies as a function of m/z show a similar linear trend on both instruments, which suggests that with appropriate collision energy adjustment, matching conditions for proteomics can be achieved. Data have been deposited in the MassIVE repository (MSV000086434).

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