Ionisation bias undermines the use of matrix‐assisted laser desorption/ionisation for estimating peptide deamidation: Synthetic peptide studies demonstrate electrospray ionisation gives more reliable response ratios

Abstract

Rationale Although mass spectrometry (MS) is routinely used to determine deamination in peptide mixtures, the effects of the choice of ionisation source have not yet been investigated. In particular, matrix‐assisted laser desorption/ionisation (MALDI) has become a popular tool with which to measure levels of glutamine deamidation in ancient proteins. Here we use model synthetic peptides to rigorously compare MALDI and electrospray ionisation (ESI). Methods We used two synthetic peptides, with glutamine (Q) in one substituted for glutamic acid (E) in the other, to investigate the suitability of MALDI and ESI sources for the assessment of deamidation in peptides using MS. We also compared measurements of the same Q‐ and E‐containing peptide mixtures using two different mass analysers (time‐of‐flight (TOF) and Fourier transform ion cyclotron resonance (FT‐ICR)). Results When standard mixtures of the Q‐ and E‐containing peptides were analysed using MALDI, under‐representation of the E‐containing peptide was observed. This observation was consistent between analyses carried out using either TOF or FT‐ICR‐MS. When the same mixtures were analysed using ESI FT‐ICR‐MS, no ionisation bias was observed. Conclusions MALDI may not be a suitable ionisation method for the determination of deamidation in peptide mixtures. However, ESI was successfully used to determine the ratio in known mixtures of Q‐ and E‐containing peptides. These preliminary observations warrant further investigation into ionisation bias when measuring deamidation in other peptide sequences.

Never boring: Non-invasive palaeoproteomics of mummified human skin

“…We assessed the authenticity of the sequences by estimating the extent of deamidation. This is a rather crude but usually effective measure of the extent of overall degradation -not necessarily of age (Schroeter and Cleland, 2016;Simpson et al, 2019Simpson et al, , 2016Welker et al, 2016). In our case, we obtained results that were internally consistent: in EVA_C (heavily handled) all collagens appeared to be "ancient" and all keratins "modern", while in EVA_M16747 (not heavily handled but with a century-long post-excavation history), all collagens and all keratins appeared "ancient".…”

Parchment Glutamine Index (PQI): A novel method to estimate glutamine deamidation levels in parchment collagen obtained from low-quality MALDI-TOF data

“…A recent study [Simpson et al, 2019] demonstrated that MALDI technique underestimates peptides with E in peptide mixtures containing Q and E substitutions that could potentially result in the systematic overestimation of q , i.e. remaining Q.…”

Parchment Glutamine Index (PQI): A novel method to estimate glutamine deamidation levels in parchment collagen obtained from low-quality MALDI-TOF data

“…A recent study (Simpson et al, 2019) demonstrated that MALDI technique underestimates peptides with E in mixtures containing Q and E substitutions that could potentially result in the systematic overestimation of q, i.e. remaining Q.…”