Mass Spec data associated with Proteomics analysis of the polyomavirus DNA replication initiation complex reveals a novel functional phosphorylated residue- IJMS 2024

Published: 3 April 2024| Version 1 | DOI: 10.17632/8srzx429ms.1
, Thomas Melendy


Polyomavirus Large T-antigen (LT) is the major viral regulatory protein that targets numerous cellular factors/pathways for cellular transformation and viral replication. LT directly recruits the cellular replication factors involved in recognition of the viral origin, origin unwinding, and primer synthesis carried out by mutual interactions between LT, DNA polymerase alpha-primase (Polprim), and single-stranded DNA binding complex, (RPA). The activities as well as protein-protein interactions of these complexes, are known to be modulated by post-translational modifications; however, modern high-sensitivity proteomic analyses of the PTMs as well as proteins associated with the three have been lacking. Using high resolution liquid chromatography tandem mass spectrometry (LC-MS/MS) of the immunoprecipitated factors (IPMS) we identified 602 PAARs for the 8 polypeptides of LT, Polprim and RPA, of which 479 had not previously been identified: 82 PAARs on SV40 LT, 305 on the Polprim heterotetrametric complex and 92 on the RPA heterotrimeric complex. Using phosphomimetic mutation of one of the novel phosphorylated aa residues, Threonine 518 detected on LT in this study, we have demonstrated a dramatic decrease in DNA replication functions of SV40 Large T- antigen both in vitro and in cell culture. The expansion of our understanding of PAAR sites provides new avenues to investigate regulation of important viral-host interactions with implications for devising targeted therapeutic strategies. The MS data includes raw data as excel files from the UB Mass Spec Facility with Sequence maps in paper. Excel documents: SV40 LT (Cont and Etoposide treated: ETO), POLA (Cont and ETO) and RPA (Cont and ETO). Following are brief explanations of all columns in the tables: Sequence - the peptide sequence identified # PSMs - how many times a spectrum has been matched to this peptide sequence ( a crude quantitative value) # Proteins - how many protein(s) this peptide can be inferred to # Protein groups - similar with above but several proteins can be grouped to one protein group if no unique peptides can be identified to discriminated one from the other Protein group accessions - Uniprot (or other protein database) accession number Modifications - PTMs identified on the peptide deltaCn - the difference of XCorr (search engine score) between the best match and the second best match (which is a match to another peptide, usually only the best match is considered as the "correct" match) phosphoRS site probabilities - Predicted probabilities of PTM localization XCorr - search engine score from Sequest HT, the higher XCorr the more confident the identification Charge, MH+, RT - basic probabilities of the peptide ion deltaM - mass difference between the theoretical and detected peptide ion # Missed cleavages - how many K/Rs left in the peptide sequence, which should be cleaved by trypsin but are not


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Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was carried out on a trapping nano-flow LC-Orbitrap MS system consisting of a Dionex Ultimate 3000 nano LC system, a Dionex Ultimate 3000 gradient micro-LC system with an WPS-3000 au-tosampler, and an Orbitrap Fusion Lumos mass spectrometer (ThermoFisher Scientific, San Jose, CA). For preparation of lyophilized LT samples, 50μL 0.5% SDS was added to each sample, and samples were sonicated for 30 sec and vortex mixed for 10 min to re-constitute protein. Protein reduction and alkylation was performed sequentially by addition of 2μL 200μM DTT and 4μL 500μM iodoacetamide (IAM), each with 45-min incubation in a covered thermomixer under 37˚C with constant shaking. Protein was precipitated. A volume of 5μL trypsin dissolved in 50mM Tris-FA (0.25μg/μL) was added to each sample, and trypsinization was performed under 37˚C overnight (~16hr) with con-stant shaking in a covered thermomixer. Trypsinization was terminated and derived peptide mixture was centrifuged at 18,000 g under 4˚C for 30 min, and supernatant was transferred to vials for analysis. A single injection of derived peptides was analyzed for each sample. A large-i.d. trapping column (300µm ID × 5 mm) was implemented prior to nano LC column (75-μm ID × 100 cm, packed with 3μm Pepmap C18) separation for high-capacity sample loading. Mobile phase A and B were 0.1% FA in 2% acetonitrile and 0.1% FA in 88% acetonitrile. The 180-min LC gradient profile was: 4−13% B for 15 min; 13% to 28% B for 110 min; 28% to 44% B for 5 min; 44% to 60% B for 5 min; 60% to 97% B for 1 min, and isocratic at 97% B for 17 min. MS was operated under data-dependent acquisition (DDA) mode, with a maximal duty cycle time of 3 sec. MS1 spectra were acquired in the m/z range 400~1,500 under 120k resolution with dynamic exclusion settings (60 sec ± 10 ppm). Precursor ions were filtered by quadrupole using a 1-Th wide window and fragmented by high energy C-trap dissociation (HCD) at a nor-malized collision energy of 35%. MS2 spectra were acquired under 15k resolution in either Orbitrap (OT) or Ion Trap (IT). Data Analysis LC-MS raw files were searched by Sequest HT (embedded in Proteome Discoverer v1.4.1.14, ThermoFisher Scientific) against SV40 LT sequence. The search parameters include: 1) Precursor ion mass tolerance: 20 ppm; 2) Fragment ion mass tolerance: 0.02 Da (OT)/0.8 Da (IT); 3) Maximal missed cleavages: 2; 4) Fixed modification: cysteine carbamidomethylation; 5) Dynamic modification: methionine oxidation, peptide N-terminal acetylation (spontaneously occurring protein modifications, labeled as O and A in the protein PTM map; serine/threonine/tyrosine (S/T/Y) phosphorylation (labeled as P in the protein PTM map); 6) Maximal modifications per peptide: 4. The acetylation of free amine groups is on any aa including Lys (K) and Arg (R) termini and occur spontaneously during experimental steps and may not be biologically meaningful unless validated.


University at Buffalo Department of Microbiology and Immunology, University at Buffalo - Downtown Campus


Bioinformatics, DNA Replication, Proteomics, Phosphorylation, Transcription Factor, Network Analysis, Polyomavirus, DNA Virus, Simian Virus 40


National Institute for Health and Care Research