Česká společnost pro hmotnostní spektrometrii

(CEEPC/IPM/CMSC 2022 - ThS-04)
Collision energy setting in proteomics and glycoproteomics: From individual species to a practical perspective

Agnes Revesz ^1 *, Laszlo Drahos ¹, Karoly Vekey ¹, Kinga Nagy ¹, Gitta Schlosser ²

1. Research Centre for Natural Sciences, Budapest, Hungary
2. Lendület Ion Mobility Mass Spectrometry Research Group, Eötvös University, Budapest, Hungary

Abstrakt

In bottom-up proteomics, the choice of collision energy in tandem mass spectrometric experiments has outstanding role since it fundamentally influences the obtained fragmentation pattern. A few years ago, we were the first to examine a direct connection between the collision energy setting and the identification confidence for a large number of individual peptides.[1] The unprecedented amount of peptide-level information, processed by our in-house developed program Serac, has provided new insights into the factors behind the overall performance of bottom-up methodologies. It allowed us to design optimized workflows, and to suggest simple approaches to quickly optimize methods on other instruments.

Recently, we have focused to the more challenging glycopeptides, where we mapped the Byonic and pGlyco search engine scores of almost 200 individual N-glycopeptides from standards as a function of collision energy settings. We found that the peptide sequence heavily influences the energy for the highest score, on top of an expected general linear trend with m/z. Our optimized workflow based on the results led to 10-50% more identified N-glycopeptides on HeLa, blood plasma, and monoclonal antibody samples.
We also investigated how the two most widely employed MS instruments (QTof and Orbitrap) should be set up such that they deliver spectra with comparable information content. To this end, energy dependent studies were performed for a large number of peptides on different instruments and similarity indices were determined between the obtained MS/MS spectra.[2]

Last, but not least, we have started a systematic investigation to unravel the impact of the ion mobility cell on the optimum collision energy at the individual peptide level.

* Korespondující autor: revesz.agnes@ttk.hu

Literatura

1. Révész Á. et al.: Mass Spectrom. Rev. 2021
2. Szabó D. et al.: J. Mass Spectrom. 2021, 56, e4693

Poděkování:

Funding from the National Research, Development and Innova-tion Office (NKFIH PD-132135, FK-138678, K-119459 and K-131762) is gratefully acknowledged.