Ultraviolet, Infrared, and High-Low Energy Photodissociation of Post-Translationally Modified Peptides

Abstract

Mass spectrometry-based methods have made significant progress in characterizing post-translational modifications in peptides and proteins; however, certain aspects regarding fragmentation methods must still be improved. A good technique is expected to provide excellent sequence information, locate PTM sites, and retain the labile PTM groups. To address these issues, we investigate 10.6 μm IRMPD, 213 nm UVPD, and combined UV and IR photodissociation, known as HiLoPD (high-low photodissociation), for phospho-, sulfo-, and glyco-peptide cations. IRMPD shows excellent backbone fragmentation and produces equal numbers of N- and C-terminal ions. The results reveal that 213 nm UVPD and HiLoPD methods can provide diverse backbone fragmentation producing a/x, b/y, and c/z ions with excellent sequence coverage, locate PTM sites, and offer reasonable retention efficiency for phospho- and glyco-peptides. Excellent sequence coverage is achieved for sulfo-peptides and the position of the SO group can be pinpointed; however, widespread SO losses are detected irrespective of the methods used herein. Based on the overall performance achieved, we believe that 213 nm UVPD and HiLoPD can serve as alternative options to collision activation and electron transfer dissociations for phospho- and glyco-proteomics. Graphical Abstract ᅟ.

A Case Study to Identify the Drug Conjugation Site of a Site-Specific Antibody-Drug-Conjugate Using Middle-Down Mass Spectrometry

“…Since MD MS might be well suited to circumvent bottom-up limitations, we thus used UVPD in a MD MS workflow to evaluate the capabilities of this activation technique to provide fragment ions characteristic of the position of the two modifications of interest (N-glycosylation and payload). This strategy avoids solubility issues related to hydrophobic drug-conjugated peptides and allows the use of UVPD, less prone to PTM fragmentation as compared to HCD (13,34). Altogether, UVPD MS/MS MD experiments of the Ides digested CBW-03-106 allowed for global amino acid sequence coverage of ~50% (44% Fc/2, 50% Fd, 62% LC), as already reported for unconjugated mAbs (Figure 2).…”

Leveraging Immonium Ions for Targeting Acyl‐Lysine Modifications in Proteomic Datasets

“…Another common fragmentation approach in the analysis of peptides and PTMs is electron transfer dissociation (ETD), [ 44–46 ] and interest in ultraviolet (UV) photodissociation (PD) is growing as well. [ 47 ] CID fragmentation yields many products, including the neutral losses that form immonium ions. ETD mostly cleaves along the Cα‐N bond of the peptide backbone, [ 44 ] thereby making immonium ion formation unlikely.…”

Proteomics and proteoforms: Bottom-up or top-down, how to use high-resolution mass spectrometry to reach the Grail

“…Commercial configurations provide a source of UV photons at the HCD cell in orbitrap, but UVPD has also been implemented successfully in an FT-ICR cell [79]. A recent study has compared and combined the effects of photoactivation by IR and UV photons to optimize the study of phospho-, glyco-and sulfo-peptides [80,81].…”