Optimisation of Milk Protein Top-Down Sequencing Using In-Source Collision-Induced Dissociation in the Maxis Quadrupole Time-of-Flight Mass Spectrometer

Optimisation of Milk Protein Top-Down Sequencing Using In-Source Collision-Induced Dissociation in the Maxis Quadrupole Time-of-Flight Mass Spectrometer

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Top-down sequencing in proteomics has come of age owing to continuous progress in LC-MS.With their high resolution and broad mass range, Quadrupole Time-of-Flight (Q-ToF) hybrid mass spectrometers equipped with electrospray ionisation source and tandem MS capability click here by collision-induced dissociation (CID) can be employed to analyse intact proteins and retrieve primary sequence information.To our knowledge, top-down proteomics methods with Q-ToF have only been evaluated using samples of relatively low complexity.Furthermore, the in-source CID (IS-CID) capability of Q-ToF instruments has been under-utilised.

This study aimed at optimising top-down sequencing of intact milk proteins to achieve the greatest sequence coverage possible from samples of increasing complexity, assessed using nine known proteins.Eleven MS/MS methods varying in their IS-CID and conventional CID parameters were tested on individual and mixed protein standards as well as raw milk samples.Top-down sequencing results from the nine most abundant proteoforms of caseins, alpha-lactalbumin and beta-lactoglubulins were compared.Nine MS/MS methods achieved more than 70% sequence coverage overall to 12n/1200 wella distinguish between allelic proteoforms, varying only by one or two amino acids.

The optimal methods utilised IS-CID at low energy.This experiment demonstrates the utility of Q-ToF systems for top-down proteomics and that IS-CID could be more frequently employed.