HLA-DR peptides from human BAL and EBV-transformed B cells
Optimization of the protocols for isolation HLA-DR (MHCII) peptides from human BAL.
Sample Processing Protocol
BAL cells and EBV-transformed B cells were lysed, total membranes were isolated and solubilized using dodecyl maltoside. HLA-DR peptide complexes were isolated by immunoprecipitation. Peptides were eluted and purified using SXC and C18 stage tips, and analyzed by LTQ Velos Orbitrap (using CID), Q Exactive or Q Exactive plus.
Data Processing Protocol
Mass lists were generated using Mascot Distiller and used to search the human complete proteome from Uniprot. The following parameters were used: precursor tolerance: 5 ppm; fragment tolerance: 0.25 Da (Velos) or 0.02 Da (Q Exactive); Enzyme specificity: none; No fixed modifications; Oxidition (M), deamidation (N/Q), pyroGlu (N-term Q) as variable modifications. Mascot scores were adjusted using Percolator.
Jimmy Ytterberg, Department of Medicine, Solna, Karolinska Institutet, Stockholm, Sweden
Roman A. Zubarev, Division of Chemistry I, Department of Medical Biochemistry & Biophysics Karolinska Institutet Stockholm, Sweden ( lab head )
Heyder T, Kohler M, Tarasova NK, Haag S, Rutishauser D, Rivera NV, Sandin C, Mia S, Malmström V, Wheelock ÅM, Wahlström J, Holmdahl R, Eklund A, Zubarev RA, Grunewald J, Ytterberg AJ. Approach for identifying HLA-DR bound peptides from scarce clinical samples. Mol Cell Proteomics. 2016 Jul 24. pii: mcp.M116.060764 PubMed: 27452731