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Molecules 2018, 23(11), 2777; https://doi.org/10.3390/molecules23112777

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

1
Department of Economic Development, Jobs, Transport and Resources, AgriBio Centre, Bundoora, Victoria 3083, Australia
2
Genedata AG, 4016 Basel, Switzerland
3
School of Applied Systems Biology, La Trobe University, Bundoora, Victoria 3083, Australia
*
Author to whom correspondence should be addressed.
Academic Editor: Paolo Iadarola
Received: 8 October 2018 / Revised: 24 October 2018 / Accepted: 25 October 2018 / Published: 26 October 2018
(This article belongs to the Special Issue Mass Spectrometric Proteomics)
Full-Text   |   PDF [2800 KB, uploaded 26 October 2018]   |  

Abstract

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 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 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. View Full-Text
Keywords: top-down proteomics; HPLC-ESI-Q-TOF MS; cow’s milk; whey proteins and caseins; tandem MS top-down proteomics; HPLC-ESI-Q-TOF MS; cow’s milk; whey proteins and caseins; tandem MS
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Vincent, D.; Mertens, D.; Rochfort, S. Optimisation of Milk Protein Top-Down Sequencing Using In-Source Collision-Induced Dissociation in the Maxis Quadrupole Time-of-Flight Mass Spectrometer. Molecules 2018, 23, 2777.

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