High Resolution Parallel Reaction Monitoring with Electron Transfer Dissociation for Middle-Down Proteomics

Abstract

In recent years, middle-down proteomics has emerged as a popular technique for the characterization and quantification of proteins not readily amenable to typical bottom-up approaches. So far, all high resolution middle-down approaches are done in data-dependent acquisition mode, using both collision-induced dissociation or electron capture/transfer dissociation techniques. Here, we explore middle-down proteomics with electron transfer dissociation using a targeted acquisition mode, parallel reaction monitoring (PRM), on an Orbitrap Fusion. As an example of a highly modified protein, we used histone H3 fractions from untreated and DMSO-treated Murine ErythroLeukemia (MEL) cells. We first determined optimized instrument parameters to obtain high sequence coverage using a synthetic standard peptide. We then setup a combined method of both MS1 scans and PRM scans of the 20 most abundant combinations of methylation and acetylation of the +10 charge state of the N-terminal tail of H3. Weak cation exchange hydrophilic interaction chromatography was used to separate the N-terminal H3 tail, primarily, by its acetylation and, to a secondary degree, by its methylation status, which aided in the interpretation of the results. After deconvolution of the highly charged ions, peaks were annotated to a minimum set of 254 H3 proteoforms in the untreated and treated samples. Upon DMSO treatment, global quantitation changes from the MS1 level show a relative decrease of 2, 3, 4, and 5 acetylations and an increase of 0 and 1 acetylations. A fragment ion map was developed to visualize specific differences between treated and untreated samples. Taken together, the data presented here show that middle-down proteomics with electron transfer dissociation using PRM is a novel, attractive method for the effective analysis and quantification of large and highly modified peptides.

Additional Information

© 2015 American Chemical Society. Received: April 23, 2015; Accepted: July 15, 2015; Publication Date (Web): July 15, 2015. The Proteome Exploration Laboratory is supported by the Gordon and Betty Moore Foundation through grant GBMF775 and the Beckman Institute and the HHMI (Orbitrap Fusion instrument). C.F. was funded by Fulbright, PROLAB (ASBMB), through a Wood-Whelan fellowship from IUBMB and from Fundação para a Ciência e Tecnologia, Portugal, with both a project grant (PTDC/MAR-BIO/2174/2012) and a postdoctoral fellowship (SFRH/BPD/79271/2011). The authors thank Dr. Andrew Alpert of PolyLC Inc. for technical advice. The authors declare no competing financial interest.

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Supplemental Material - ac5b01542_si_003.pdf

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