Huge shoutout to Drs. Si Wu and Kellye Cupp-Sutton who were an enormous help in our first top-down analysis! #TeamMassSpec, #senescence, #aging, #geroscience, #proteomics, #TopDownProteomics n/n

A key observation is the definition of SASP highly depends on control conditions. We propose that comparing with multiple controls representing different 'non-senescent' states (e.g. proliferating cells, quiescent cells,..) are best to identify robust senescence-specific proteins. 5/n

Several proteoforms emerged as unique to senescent cells versus either control condition. Notably, all multi-phosphorylated forms of HMGA2 were elevated in the SASP. Perhaps due to P-Ser flanking the nuclear localization sequence, similarly to the related HMGB1 protein, a well known SASP. 4/n

Topdown proteomics enabled us to identify 52 proteoforms mapping to 38 known SASP proteins previously reported in our bottom-up DIA SASP Atlas study. Additionally, we identified 41 proteoforms mapping to 29 proteins not previously reported. 3/n

We performed quantitative top-down secretomics on senescent, proliferating and quiescent cells to identify senescence-specific proteoforms. We used a combination of ToPIC suite (for IDs) and Biopharma Finder (for the areas of mass features) and combined the outputs for proteoform quant. 2/n

Nice congrats! I was wondering what to call BlueSky tutorials

@bradolinger.bsky.social called it “blueprint” and I liked that…

Thanks to all who contributed to the study! Including support from the NIA Intramural Research Program, NIH SenNet Common Fund Grant, and Hevolution Foundation. Thanks to the Seer team who were extremely responsive and helpful.

6/6

We hope this workflow enables other labs to perform comprehensive proteomic studies in mice, where it was not previously possible due to volume and dynamic range limitations.

#Proteomics #MassSpec #AgingResearch #Geroscience #Senescence #Biomarkers #Proteograph #bioRxiv #Olink #QEHF

5/n

We demonstrate the method's potential to detect aging /phenotype associated changes in serum. We observed completely different changes in blood at several ‘old’ ages, such as 24 months (old) and 30month (geriatric), reinforcing the importance of including multiple timepoints in aging studies

4/n

MS studies notably miss cytokines. A targeted mouse cytokine panel (O-link) on just 1 uL serum enabled us to quantify an additional 39 proteins not detected by MS (e.g. IL6), suggesting strong complimentary to the MS workflow. As expected, the majority of the cytokines increased with age.

3/n

Led by @bradolinger.bsky.social and Amit Dey – in 20uL serum we detected nearly 4000 proteins using the Proteograph workflow (4+ fold improvement) & demonstrated its consistency and accuracy w/ an 'LFQ-Bench-like' study. All on the Q-Exactive HF, so no need for the new fancy stuff.

2/n