A targeted approach for the synthesis of multi-phosphorylated peptides: a tool for studying the role of phosphorylation patterns in proteins
Authors: Mamidi Samarasimhareddy
Daniel Mayer
Norman Metanis
Assaf Friedler
Mattan Hurevich
Dmitry Veprintsev
Journal: Organic & Biomolecular Chemistry (2019)
Discipline: CHEMISTRY
Keywords: Protein phosphorylation barcodes; multiphosphopeptide libraries; microwave assisted synthesis; C-terminus of rhodopsin; phosphorylated amino acids
Affiliations: Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel, Laboratory of Biomolecular Research, Paul Scherrer Institute, 5232 Villigen PSI, Switzerland and Department of Biology, ETH Zürich

Abstract:  Protein phosphorylation barcodes, clusters of several phosphorylation sites within a short unfolded region, control many cellular processes. Existing biochemical methods used to study the roles of these barcodes suffer from low selectivity and provide only qualitative data. Chemically synthesized multiphosphopeptide libraries are selective and specific, but their synthesis is extremely difficult using the current peptide synthesis methods. Here we describe a new microwave assisted approach for synthesizing a library of multiphosphopeptides, using the C-terminus of rhodopsin as a proof of concept. Our approach utilizes multiple protocols for synthesizing libraries of multiphosphopeptides instead of the inefficient single protocol methods currently used. Using our approach we demonstrated the synthesis with up to seven phosphorylated amino acids, sometimes next to each other, an accomplishment that was impractical before. Synthesizing the Rhodopsin derived multiphosphopeptide library enabled dissecting the precise phosphorylation barcode required for the recruitment, activation and modulation of the conformation of Arrestin. Since phosphorylation barcodes modulate the activity of hundreds of GPCRs, synthesizing libraries of multiphosphopeptides is the method of choice for studying their molecular mechanisms of action. Our approach provides an invaluable tool for evaluating how protein phosphorylation barcodes regulate their activity. 

Retracted: No

PubPeer comments:
 The authors present a surprising advancement in phosphopeptide synthesis with only modest changes to standard SPPS methodology in this article. Generally, coupling three phosphorylated amino acids within a single peptide would be quite a feat but here they are able to achieve seven. It would be greatly appreciated if the authors could address several comments and concerns I have here: 1) In the discussion, it is mentioned that repeat coupling reactions with pAA were performed to overcome steric hinderance - however it is not clear why repeating a coupling reaction helps. The reaction already contains five equivalents of pAA, and assuming only 50% of the pAA was coupled before the next reaction cycle- that would leave ~4.5 equiv of pAA within the reaction. Wouldn t it be more reagent efficient to just double or triple the reaction time in the first coupling cycle to achieve quantitative coupling? 2) The conclusion claims that multiple phosphorylated peptides were synthesized in  good yields , but there isn t a single yield quantified throughout the paper. What are the yields of the peptides with and without optimization? 3) Did the authors observe any benzylation as a side-product during peptide cleavage? This is something that can occur, and I wouldn t be entirely surprised with seven phospho residues. 4) There are several issues with the data that I think should be addressed: Figure 4A and 4B - 4A shows the chromatogram extending beyond the 0 min mark. The deconvoluted mass shown in 4B does not align correctly with the x-axis. The mass is 2598.46 but the peak is past the 2,600 (m z or Da) tick mark. ![file] Figure S1 - Something is really unusual about both the deconvoluted mass and the chromatogram. I ve highlighted some of the issues with red boxes. I ve never seen peaks that shift in the negative x-axis direction and create such a distorted effect. ![file] Figure S6 - Stated mass doesn t align with presented x-axis. ![file] Figure S12 - Stated mass doesn t align with presented x-axis. The mass peak is above 2550 Da. ![file] Figure S15 - Same as above. Also the resolution is very different in this analysis or the sample is not pure. ![file] Figure S21 - same issue as above. ![file] Figure S30 - multiple issues here. Similar to S1, the peaks appear almost drawn with unusual inflections. There are also peaks that appear to have been cut out - based on discontinuity of the signal near the x-axis. ![file] ,