Diversity Matters: Optimal Collision Energies for Tandem Mass Spectrometric Analysis of a Large Set of N-Glycopeptides

[Image: see text] Identification and characterization of N-glycopeptides from complex samples are usually based on tandem mass spectrometric measurements. Experimental settings, especially the collision energy selection method, fundamentally influence the obtained fragmentation pattern and hence the confidence of the database search results (“score”). Using standards of naturally occurring glycoproteins, we mapped the Byonic and pGlyco search engine scores of almost 200 individual N-glycopeptides as a function of collision energy settings on a quadrupole time of flight instrument. The resulting unprecedented amount of peptide-level information on such a large and diverse set of N-glycopeptides revealed that the peptide sequence heavily influences the energy for the highest score on top of an expected general linear trend with m/z. Search engine dependence may also be noteworthy. Based on the trends, we designed an experimental method and tested it on HeLa, blood plasma, and monoclonal antibody samples. As compared to the literature, these notably lower collision energies in our workflow led to 10–50% more identified N-glycopeptides, with higher scores. We recommend a simple approach based on a small set of reference N-glycopeptides easily accessible from glycoprotein standards to ease the precise determination of optimal methods on other instruments. Data sets can be accessed via the MassIVE repository (MSV000089657 and MSV000090218).