immunoglobulin light chains and attribution to a germline gene-based - PowerPoint PPT Presentation

A novel method for proteomic analysis of immunoglobulin light chains and attribution to a germline gene-based family Alessandra Tiengo Department of Computer Engineering and Systems Science UNIVERSITÀ DI PAVIA

Amyloidosis Tissue deposition of misfolded Amyloidosis proteins as amyloid fibrils Deposition of monoclonal free AL Amyloidosis immunoglobulin light chains (LC) Subgroups or families Alessandra Tiengo NETTAB, Pavia, 2011 IIT@SEMM, Milan, 2011 Angelo Nuzzo

Subgroup assignment Proteomic approach (PMF) Gene-based approach 1. MS experiments 1. Coding mRNA (bone marrow plasma cells) 2. Database of theoretical peptides (e.g. Swiss-Prot, IMGT) 2. cDNA sequencing 3. Comparison between theoretical and experimental spectra Requires bone marrow (Invasive and long procedure) Variable Regions Alessandra Tiengo NETTAB, Pavia, 2011 IIT@SEMM, Milan, 2011 Angelo Nuzzo

Subgroup assignment 1. Database 2. Peptide 3. Subgroup processing analysis identification Alessandra Tiengo NETTAB, Pavia, 2011 IIT@SEMM, Milan, 2011 Angelo Nuzzo

Database processing 2. Peptide 1. Database 3. Subgroup processing analysis identification IMGT database In silico digestion MCs and PTMs New DB FR coverage LC subgroup Alessandra Tiengo NETTAB, Pavia, 2011 IIT@SEMM, Milan, 2011 Angelo Nuzzo

Peptide analysis 2. Peptide 1. Database 3. Subgroup processing analysis identification Peptide frequency in the subgroup ASCII file Median FR coverage of the peptide m/z Freq. FR cov. Class 801 0.1 0.3 LC1 Output: ASCII file containing the 1100 0 0.8 LC10 1398 0.5 0 LC1 peptide mass, the frequency, the 2100 0 0 LC3 2168 0.2 0.1 LC1 median FR coverage and the subgroup … … … … Alessandra Tiengo NETTAB, Pavia, 2011 IIT@SEMM, Milan, 2011 Angelo Nuzzo

Subgroup identification 2. Peptide 1. Database 3. Subgroup processing analysis identification Peak list Comparison between MS spectra and ASCII file peptides in the ASCII file (tolerance window in Da or ppm) m/z Int Subgroup identification m/z Freq. FR cov. Class 801 25.0 N  801 0.1 0.3 LC1   Score ( S ) Int Freq 1100 39.0 1100 0 0.8 LC10 1 n m ( S )  n 1 1400 15.0 1398 0.5 0 LC1 N  2100 0 0 LC3    1406 100.0 Score ( S ) Int Freq FR 2168 0.2 0.1 LC1 2 n m ( S ) m ( S )  1512 24.0 n 1 … … … … N  … …   Score ( S ) Int FR 3 n m ( S )  n 1 N matching peaks Alessandra Tiengo NETTAB, Pavia, 2011 IIT@SEMM, Milan, 2011 Angelo Nuzzo

Results 20 MS spectra (10 patients) IMGT database (3178 human proteins with annotated LC subgroup) 1 MC PTMs: CAM (C), oxydation (M), pyro-Glu (Q), deletion of first amino acid Tolerance mass: 0.3 Da Comparison between the scoring functions and MsPI (PMF approach) cDNA sequencing assigment considered as correct Alessandra Tiengo NETTAB, Pavia, 2011 IIT@SEMM, Milan, 2011 Angelo Nuzzo

Thanks! Alessandra Tiengo NETTAB, Pavia, 2011 IIT@SEMM, Milan, 2011 Angelo Nuzzo