Top-Down Proteomics Enables Comparative Analysis of Brain Proteoforms Between Mouse Strains

Abstract

Over the past decade, advances in mass spectrometry-based proteomics have accelerated brain proteome research aimed at studying the expression, dynamic modification, interaction and function of proteins in the nervous system that are associated with physiological and behavioral processes. With the latest hardware and software improvements in top-down mass spectrometry, the technology has expanded from mere protein profiling to high-throughput identification and quantification of intact proteoforms. Murine systems are broadly used as models to study human diseases. Neuroscientists specifically study the mouse brain from inbred strains to help understand how strain-specific genotype and phenotype affect development, functioning, and disease progression. This work describes the first application of label-free quantitative top-down proteomics to the analysis of the mouse brain proteome. Operating in discovery mode, we determined physiochemical differences in brain tissue from four healthy inbred strains, C57BL/6J, DBA/2J, FVB/NJ, and BALB/cByJ, after probing their intact proteome in the 3.5-30 kDa mass range. We also disseminate these findings using a new tool for top-down proteomics, TDViewer and cataloged them in a newly established Mouse Brain Proteoform Atlas. The analysis of brain tissues from the four strains identified 131 gene products leading to the full characterization of 343 of the 593 proteoforms identified. Within the results, singly and doubly phosphorylated ARPP-21 proteoforms, known to inhibit calmodulin, were differentially expressed across the four strains. Gene ontology (GO) analysis for detected differentially expressed proteoforms also helps to illuminate the similarities and dissimilarities in phenotypes among these inbred strains.

Mapping Microproteins and ncRNA-Encoded Polypeptides in Different Mouse Tissues

“…The results indicated that microproteins in different tissues were mostly related to tissue-specific functions ( Figure 5D ). For example, brain tissue has many microproteins related to nerves and hormones, which is consistent with previous research findings ( Marcus et al, 2004 ; Davis et al, 2018 ). Splenic tissue was rich in immunity-related microproteins, which is consistent with previous research ( Hu et al, 2018 ; Ma et al, 2019 ).…”

Identification and Quantification of Proteoforms by Mass Spectrometry

“…Labeling reagents are not needed in LFQ, so it can be applied to samples not grown in cell culture, such as patient tissue samples. LFQ has been applied to several larger scale studies of proteoform abundance changes across two conditions . The Kelleher lab first demonstrated this technique using yeast mutant versus wild‐type strains and quantified over 800 proteoforms.…”

Neurodegenerative Proteinopathies in the Proteoform Spectrum—Tools and Challenges

“…Top-down proteomics is becoming increasingly applicable for the study of eukaryotic proteome and proteoforms [ 92 , 94 ]. It is also being used for the study of native prion-like proteins.…”