MassIVE MSV000082070

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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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Description

Top-down sequencing in proteomics has come of age owing to continuous progress brought to LC-MS. The quality and thoroughness of the results depend on the platform used. The latest generation of mass spectrometers displays unprecedented resolving power, very fast scanning rates, and several fragmentation modes. Unfortunately these instruments are very expensive, therefore out of reach for most laboratories. With their high resolution and broad mass range, quadrupole Time-of-Flight (Q-ToF) hybrid mass spectrometers equipped with electrospray ionisation (ESI) source and tandem MS capability by collision-induced dissociation (CID) offer a valuable alternative to analyse intact proteins and retrieve primary sequence information. As far as we know, top-down proteomics methods with such systems have only been evaluated using samples of relatively low complexity. The in source CID (IS-CID) capability of the Q-ToF instruments has been underutilised. This study aimed at optimising top-down sequencing results on intact milk proteins to achieve the greatest sequence coverage possible from samples of increasing levels of complexity. Eleven MS/MS methods varying in their IS-CID and conventional CID parameters were tested on individual and mixed protein standards and raw milk samples from two main cow breeds. 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 percents sequence coverage overall to distinguish between allelic proteoforms, varying only by one or two AAs. Full 100 percent sequence information was obtained on bLGs. Two methods (Methods 8 and 10) employed high IS-CID energy failed to distinguish between allelic variations. The most optimal methods (Methods 4 and 12) utilised IS-CID on its own at low energy. Under optimal analytical conditions, sample complexity did not impact the quality and reproducibility of the results. This experiment demonstrates that Q-ToF systems remain suitable for top-down proteomics and that IS-CID should be more frequently utilised. doi:10.3390/molecules23112777 [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: Top-down proteomics, HPLC-ESI-Q-TOF MS, cow milk, whey proteins and caseins, tandem MS ; Genedata Refiner software

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Principal Investigators:
(in alphabetical order) 		Delphine Vincent, DEDJTR, Government State of Victoria, Australia
Submitting User: delphine_1_2
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