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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.
Highlights
Top-down proteomics, a term invented by Kelleher and colleagues 20 years ago [1], describes the analysis of intact proteins, either in their native form or more often in a denatured state, which allows for a characterisation of proteoforms as comprehensively as possible
The most important technological advance was the coupling of the soft ionisation technique, electrospray ionisation (ESI), to a mass spectrometer and the production of gas phase ions from large molecules [3], which led to its first application to Molecules 2018, 23, 2777; doi:10.3390/molecules23112777
Further developments in intact protein analysis quickly followed with the introduction of complex multistage mass analysers of high resolution such as linear ion trap (LIT), quadrupole time-of-flight (Q-ToF), Fourier transform (FT)
Summary
Top-down proteomics, a term invented by Kelleher and colleagues 20 years ago [1], describes the analysis of intact proteins, either in their native form or more often in a denatured state, which allows for a characterisation of proteoforms as comprehensively as possible. Coined in 2014, the term proteoform “designates all of the different molecular forms in which the protein product of a single gene can be found, encompassing all forms of genetic variation, alternative splicing of RNA transcripts, and post-translational modifications (PTMs)” [2]. ESI generates multiply-charged protein ions of low m/z, thereby allowing the analysis of very large molecules, even on an MS platform with a limited mass range. Further developments in intact protein analysis quickly followed with the introduction of complex multistage mass analysers of high resolution such as linear ion trap (LIT), quadrupole time-of-flight (Q-ToF), Fourier transform (FT)
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