Thread by @Ben_Leo_Parker

Mass spec-based #phosphoproteomics has revolutionized the systems-wide analysis of phosphorylation. A 🧵 of some great developments for global analysis of kinase/phosphatase regulation and substrate phosphorylation…1/23

First step is enrichment. PAC, IMAC and MOAC are the most popular. In 1986, Andersson and Porath were one of the first to show that Fe3+ could bind phosphoamino acids sciencedirect.com/science/articl…
In 1997, Ikeguchi & Nakamura first used TiO2 2/23 link.springer.com/article/10.211…

In 2002, Forest White and co-workers further developed IMAC to identify 383 phosphosites from yeast representing one of the early phosphoproteome analyses. 3/23 nature.com/articles/nbt03…

Significant further developments for phosphopeptide enrichment were developed in 2004/5 Kuroda et al., Pinkse & @hecklab, and Larsen et al transforming the enrichments with extremely high selectivity. 4/24 jstage.jst.go.jp/article/analsc… pubs.acs.org/doi/10.1021/ac… linkinghub.elsevier.com/retrieve/pii/S…

Another first notable mention to detect phosphopeptides without enrichment was the use of precursor ion-scanning by Carr and co-workers in 1996 5/23 sciencedirect.com/science/articl…

Phospho-tyrosine represents another layer of difficulty owing to extreme low abundance. Antibody-based enrichment has dominated with early work developed by Salomon et al in 2003 to identify 64 tyrosine phosphorylation sites from mammalian cells. 6/23 pnas.org/doi/10.1073/pn…

Mid to late 2000’s saw some big gains in characterization; >2K nuclear sites by @GygiLab, pnas.org/doi/10.1073/pn…, >6K sites following EGF from @jespervolsen & @labs_mann doi.org/10.1016/j.cell…, >5.6K sites from mouse liver by @juditvr and @GygiLab 7/23 pnas.org/doi/10.1073/pn…

Collisional dissociation results in fragmentation of phosphate due to lower activation barriers (~40 vs <20 kcal/mol for Ccarbonyl-N v phosphoester bond). In 2004, @Coon_Labs showed a pseudo-MS3 approach (MSA) to trigger activation of neutral loss. 8/23 pubs.acs.org/doi/10.1021/ac…

Alternative fragmentation with ETD/ECD the most popular. Early studies from Hunt >1.2K sites from yeast pnas.org/doi/full/10.10…, @dlswaney, @Coon_Labs >10K sites from ES cells pnas.org/doi/full/10.10…, & Pandey etal >1.4K sites from 293 cells 9/23 pnas.org/doi/epdf/10.10…

While we are talking about ETD, we must mention supplemental/concurrent activation. The most beautiful phosphopeptide spectra from @hecklab with EThcD pubs.acs.org/doi/10.1021/pr…, and @riley_nm1 & @Coon_Labs with AI-ETD 10/23 pubs.acs.org/doi/10.1021/ac…

Despite these develops collisional dissociation is still the most popular owing to speed and efficiency. The debate on resonance-CID mcponline.org/article/S1535-… or beam-CID pubs.acs.org/doi/10.1021/pr… probably needs to be explored further on the latest instruments. 11/24

Localization to a precise amino acid is very important for functional assessment. Early work by @GygiLab presented Ascore nature.com/articles/nbt12…, MD-score from @savitski_lab and @kusterlab mcponline.org/article/S1535-… and PhoshoRS by @Karl_Mechtler pubs.acs.org/doi/10.1021/pr… 12/23

Other, diagnostic fragment ions include immonium ions e.g pubs.acs.org/doi/10.1021/ac… & nature.com/articles/nmeth…, and site-specific x-ions 13/23 pubs.acs.org/doi/10.1021/pr…

There’s an elephant in the room. Gas-phase rearrangement @gurugav69 pubs.acs.org/doi/10.1021/ac… and although low eg @hecklab mcponline.org/article/S1535-… and @GygiLab pubs.acs.org/doi/10.1021/pr…, it occurs! doi.org/10.1002/pmic.2… and @mjmaccoss & @briansearle

https://twitter.com/mjmaccoss/status/1540411970590978048

14/23

Keeping with this theme of localization, Arg and His phosphorylation have pronounced challenges e.g work by Jensen et al pubs.acs.org/doi/10.1021/ac…, @Karl_Mechtler doi.org/10.1002/pmic.2… and @hecklab nature.com/articles/s4159… with the latter particularly interesting 😲. 15/23

Quick mention to deep dives. >38K sites @EdHuttlin & @GygiLab doi.org/10.1016/j.cell…, >38K sites @TheCoxLab and @labs_mann doi.org/10.1016/j.celr… with validation using synthetic library by @kusterlab nature.com/articles/nbt.2…, >37K sites by @SeanJHumphrey & @davidjamesoz. 16/23

Fast phosphoproteomes! More recently, >8K sites in 15min by @fmeierX biorxiv.org/content/10.110… >14K sites in 15min by @jespervolsen nature.com/articles/s4146… and >35K sites in 21min by @labs_mann biorxiv.org/content/10.110…. Wow! 17/23

A couple of targeted approaches. Plug for my analysis using DIA with @GuangYa15456338 @davidjamesoz science.org/doi/10.1126/sc… and the plug-and-play assays from @briansearle @juditvr nature.com/articles/nmeth…. SureQuant is also a growing approach 18/23 doi.org/10.1158/0008-5…

We have only spoken about ID’s, @jespervolsen presented an excellent comparison of the various quantification approaches. Spoiler, for their experimental design, MS2-based TMT identified the greatest number of regulated sites! 19/23 nature.com/articles/s4146…

Assessing kinome actvity. KAYAK by @GygiLab pnas.org/doi/full/10.10… & nature.com/articles/nbt.1…. The reduced-representation phosphoproteome (P100) developed by Jaffe and coworkers was able to profile the activity of kinases in >3000 of samples! 20/23 doi.org/10.1016/j.cels…

Assessing kinase:substrate relationships (KSRs), Tao developed the Kinase Assay Linked with Phosphoproteomics (KALIP) pnas.org/doi/full/10.10…. Another cool assay I love is the whole-cell in vitro kinase assay developed by knight etal 21/23 …eletalmusclejournal.biomedcentral.com/articles/10.11…

The community still has a huge amount of work to establish KSRs. Heroic efforts are being made by @CantleyLab, @mbyaffe and Turk and many others 20/23 biorxiv.org/content/10.110…

Finally, functional characterizations can be predicted e.g by @pedrobeltrao nature.com/articles/s4158… and characterized en masse with @TypasLab and @savitski_lab nature.com/articles/s4158… 22/23

I hope you have enjoyed this tread. Would love to hear your further developments. As beautifully summarized by @elise_needham @SeanJHumphrey (check out Supp videos!), there is still a massive amount of exploration left in the dark phosphoproteome!!! 23/23 science.org/doi/10.1126/sc…