Rinehart Human Serine Phosphopeptide Library
(Pooled Libraries #111704, #111705, #111706, #111707, #111708)

• Purpose:

  The Rinehart lab has synthesized 110,139 unique DNA sequences corresponding to every previously observed instance of human serine phosphorylation. The phosphorylation sites (“phosphosites”) consist of a central phosphoserine site flanked by 15 amino acids on either side occurring within the parent protein.

  The mode #1 library can be used for purification of protein libraries or to identify candidate human substrates of kinases of interest by mass spectrometry.

  Mode #2 libraries can be used in Hi-P experiments for screening phosphorylation-dependent protein-protein interactions by fluorescence-activated cell sorting (FACS).

• Vector Backbone: Pooled Library 111704 (Mode #1) - modified pNAS1
  Pooled Library 111705 (Mode #2) - pNAS1b split mCherry 14-3-3β
  Pooled Library 111706 (Mode #2) - pNAS1b split mCherry 14-3-3σ
  Pooled Library 111707 (Mode #2) - pNAS1b split mCherry NEDD4 WW2
  Pooled Library 111708 (Mode #2) - pNAS1b split mCherry NEDD4-2 WW2

• Depositing Labs: Jesse Rinehart
• Publication: Barber et al. Nat Biotechnol. 2018 Aug;36(7):638-644. doi: 10.1038/nbt.4150. Epub 2018 Jun 11.

Library Details

• Species: Human
• Phosphopeptide sequences: 110,139

Library Shipping

This library is delivered as suspended DNA in a microcentrifuge tube on blue ice. The tube's contents will not necessarily be frozen. For best results, minimize freeze-thaws.

NOTE: 1 µL of the library can be electroporated into cells for an experiment. Sufficient sample is provided for several experiments and amplification of the library.

• Volume: ∼10 µL
• Concentration: 100 ng/µL

Resource Information

• Protocols:
  • Library Amplification (17 KB)
  • PCR amplification (13 KB)
• Depositor Data:
  • Phosphosite sequences (11 MB)
  • Nucleotide Reference to Validate Amplifications (11.7 MB)
• Terms and Licenses:
  • UBMTA
  • Takara Bio Limited Use Label License (formerly Clontech)

Depositor Comments

Please note that all libraries can be used as-is for experiments or for subsequent amplifications. Please see the associated protocols for additional information.

Using the recoded strain of E. coli C321.ΔA with the optimized phosphoserine orthogonal translation system (SepOTSλ), the Rinehart lab demonstrated unambiguous site-specific incorporation of phosphoserine in >36,000 phosphosites by tandem mass spectrometry ( Barber et al, Nat. Biotech. 2018).

This phosphosite library was generated in a single mixed pool by expressing and purifying the phosphosite library as GST-fusion peptides (“mode #1” expression). The library is useful for laboratories performing phosphoproteomic experiments in need of reference spectra or phosphopeptide standards for quantitative assessments. The mode #1 phosphosite library can be generated with either phosphoserine using SepOTSλ or serine using supD tRNA at the central position.

When generated with serine at the central position, the mode #1 library can also be used to identify candidate human substrates of kinases of interest by mass spectrometry in a technique called serine-oriented library/kinase-library reactions (SERIOHL-KILR, Barber et al, Biochemistry 2018).

The phosphosite DNA library can be introduced into a second expression vector encoding a split mCherry fluorescent reporter, enabling the detection of phosphorylation-dependent protein-peptide interactions (“mode #2” expression, Barber et al, Nat. Biotech. 2018). The phosphosite library is fused to the N-terminal portion of split mCherry, while a phosphobinding domain is fused to the C-terminal mCherry fragment. Upon interaction between the phosphobinding domain and the phosphosite, mCherry fluorescence is restored, so interactions can be discovered by fluorescence-activated cell sorting (FACS). The mode #2 phosphosite library is available with four different phosphobinding domains (14-3-3β, 14-3-3σ, the WW2 domain of NEDD4, and the WW2 domain of NEDD4-2).