shotgun-proteomics-sample-processing-automated-an-open-source-lab

Shotgun Proteomics Sample Processing Automated by an Open-Source Lab Robot

Mass spectrometry-based shotgun proteomics experiments require multiple sample preparation steps, including enzymatic protein digestion and clean-up, ...

University of Colorado Anschutz Medical Campus

Transcript abundance and protein abundance show modest correlation in many biological models, but how this impacts disease signature discovery in omics experiments is rarely explored. Here we report an integrated omics approach, incorporating measurements of transcript abundance, protein abundance, and protein turnover to map the landscape of proteome remodeling in a mouse model of pathological cardiac hypertrophy. Analyzing the hypertrophy signatures that are reproducibly discovered from each omics data type across six genetic strains of mice, we find that the integration of transcript abundance, protein abundance, and protein turnover data leads to 75% gain in discovered disease gene candidates. Moreover, the inclusion of protein turnover measurements allows discovery of post-transcriptional regulations across diverse pathways, and implicates distinct disease proteins not found in steady-state transcript and protein abundance data. Our results suggest that multi-omics investigations of proteome dynamics provide important insights into disease pathogenesis in vivo.

Integrated omics dissection of proteome dynamics during cardiac remodeling.

Small molecule inhibitors of the acetyl-histone binding protein BRD4 have been shown to block cardiac fibrosis in preclinical models of heart failure (HF). However, since the inhibitors target BRD4 ubiquitously, it is unclear whether this chromatin reader protein functions in cell type-specific manner to control pathological myocardial fibrosis. Furthermore, the molecular mechanisms by which BRD4 stimulates the transcriptional program for cardiac fibrosis remain unknown.

Dynamic Chromatin Targeting of BRD4 Stimulates Cardiac Fibroblast Activation.

Working the literature harder: what can text mining and bibliometric analysis reveal?

The protein-level translational status and function of many alternative splicing events remain poorly understood. We use an RNA sequencing (RNA-seq)-guided proteomics method to identify protein alternative splicing isoforms in the human proteome by constructing tissue-specific protein databases that prioritize transcript splice junction pairs with high translational potential. Using the custom databases to reanalyze ∼80 million mass spectra in public proteomics datasets, we identify more than 1,500 noncanonical protein isoforms across 12 human tissues, including ∼400 sequences undocumented on TrEMBL and RefSeq databases. We apply the method to original quantitative mass spectrometry experiments and observe widespread isoform regulation during human induced pluripotent stem cell cardiomyocyte differentiation. On a proteome scale, alternative isoform regions overlap frequently with disordered sequences and post-translational modification sites, suggesting that alternative splicing may regulate protein function through modulating intrinsically disordered regions. The described approach may help elucidate functional consequences of alternative splicing and expand the scope of proteomics investigations in various systems.

Splice-Junction-Based Mapping of Alternative Isoforms in the Human Proteome.

The heart is sensitive to oxidative damage but a global view on how the cardiac proteome responds to oxidative stressors has yet to fully emerge. Using quantitative tandem mass spectrometry, we assessed the effects of acute exposure of the oxidative stress inducer paraquat on protein expression in mouse hearts. We observed widespread protein expression changes in the paraquat-exposed heart especially in organelle-containing subcellular fractions. During cardiac response to acute oxidative stress, proteome changes are consistent with a rapid reduction of mitochondrial metabolism, coupled with activation of multiple antioxidant proteins, reduction of protein synthesis and remediation of proteostasis. In addition to differential expression, we saw evidence of spatial reorganizations of the cardiac proteome including the translocation of hexokinase 2 to more soluble fractions. Treatment with the antioxidants Tempol and MitoTEMPO reversed many proteomic signatures of paraquat but this reversal was incomplete. We also identified a number of proteins with unknown function in the heart to be triggered by paraquat, suggesting they may have functions in oxidative stress response. Surprisingly, protein expression changes in the heart correlate poorly with those in the lung, consistent with differential sensitivity or stress response in these two organs. The results and data set here could provide insights into oxidative stress responses in the heart and avail the search for new therapeutic targets.

Proteomic signatures of acute oxidative stress response to paraquat in the mouse heart.

Alternative splicing is prevalent in the heart and implicated in many cardiovascular diseases, but not every alternative transcript is translated and detecting non-canonical isoforms at the protein level remains challenging. Here we show the use of a computation-assisted targeted proteomics workflow to detect protein alternative isoforms in the human heart. We build on a recent strategy to integrate deep RNA-seq and large-scale mass spectrometry data to identify candidate translated isoform peptides. A machine learning approach is then applied to predict their fragmentation patterns and design protein isoform-specific parallel reaction monitoring detection (PRM) assays. As proof-of-principle, we built PRM assays for 29 non-canonical isoform peptides and detected 22 peptides in a human heart lysate. The predictions-aided PRM assays closely mirrored synthetic peptide standards for non-canonical sequences. This approach may be useful for validating non-canonical protein identification and discovering functionally relevant isoforms in the heart.

Computation-assisted targeted proteomics of alternative splicing protein isoforms in the human heart.

Diastolic dysfunction (DD) is associated with the development of heart failure and contributes to the pathogenesis of other cardiac maladies, including atrial fibrillation. Inhibition of histone deacetylases (HDACs) has been shown to prevent DD by enhancing myofibril relaxation. We addressed the therapeutic potential of HDAC inhibition in a model of established DD with preserved ejection fraction.

Article	Year
Integrated omics dissection of proteome dynamics during cardiac remodeling. Nature communications\| PubMed ID: 29317621	2018
Dynamic Chromatin Targeting of BRD4 Stimulates Cardiac Fibroblast Activation. Circulation research\| PubMed ID: 31409188	2019
Working the literature harder: what can text mining and bibliometric analysis reveal? Expert review of proteomics\| PubMed ID: 31822126	2019
Splice-Junction-Based Mapping of Alternative Isoforms in the Human Proteome. Cell reports\| PubMed ID: 31825849	2019
Proteomic signatures of acute oxidative stress response to paraquat in the mouse heart. Scientific reports\| PubMed ID: 33116222	2020
Computation-assisted targeted proteomics of alternative splicing protein isoforms in the human heart. Journal of molecular and cellular cardiology\| PubMed ID: 33549679	2021
HDAC Inhibition Reverses Preexisting Diastolic Dysfunction and Blocks Covert Extracellular Matrix Remodeling. Circulation\| PubMed ID: 33682427	2021

Maggie P. Y. Lam

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