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Charge site manipulation to enhance top-down fragmentation efficiency

ABSTRACT: In recent years, top-down mass spectrometry has become a widely used approach to study proteoforms; however, improving sequence coverage remains an important goal. Here, two different proteins, α-synuclein and bovine carbonic anhydrase, were subjected to top-down collision-induced dissociation (CID) after electrospray ionisation. Two high-boiling solvents, DMSO and propylene carbonate, were added to the protein solution in low concentration (2%) and the effects on the top-down fragmentation patterns of the proteins were systematically investigated. Each sample was measured in triplicate, which revealed highly reproducible differences in the top-down CID fragmentation patterns in the presence of a solution additive, even if the same precursor charge state was isolated in the quadrupole of the instrument. Further investigation supports the solution condition-dependent selective formation of different protonation site isomers as the underlying cause of these differences. Higher sequence coverage was often observed in the presence of additives, and the benefits of this approach became even more evident when datasets from different solution conditions were combined, as increases up to 35% in cleavage coverage were obtained. Overall, this approach therefore represents a promising opportunity to increase top-down fragmentation efficiency.

INSTRUMENT(S): Synapt MS

ORGANISM(S): Homo Sapiens (human) Bos Taurus (bovine)

SUBMITTER: Tanja Habeck

LAB HEAD: Frederik Lermyte

PROVIDER: PXD039993 | Pride | 2023-06-27

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	CA_32__rawdata.zip	Other
	MASHresult_CID_CA_32__DMSO.xlsx	Xlsx
	MASHresult_CID_CA_32__PC.xlsx	Xlsx
	MASHresult_CID_CA_32__noadditive_.xlsx	Xlsx
	MASHresult_CID_aSN_10__DMSO.xlsx	Xlsx

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Project 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

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Charge site manipulation to enhance top-down fragmentation efficiency

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