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

Jan 09, 2023 125 likes •235 views

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 UNIVERSIT DI PAVIA Amyloidosis Tissue deposition

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UNIVERSITÀ DI PAVIA

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

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

Amyloidosis

Tissue deposition of misfolded proteins as amyloid fibrils Amyloidosis

AL Amyloidosis Deposition of monoclonal free immunoglobulin light chains (LC) Subgroups

  • r families
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Angelo Nuzzo IIT@SEMM, Milan, 2011 NETTAB, Pavia, 2011 Alessandra Tiengo

Subgroup assignment

  • 2. cDNA sequencing
  • 1. Coding mRNA

(bone marrow plasma cells) Requires bone marrow (Invasive and long procedure)

Proteomic approach (PMF)

  • 3. Comparison between

theoretical and experimental spectra

  • 2. Database of theoretical

peptides (e.g. Swiss-Prot, IMGT)

  • 1. MS experiments

Variable Regions

Gene-based approach

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

Subgroup assignment

  • 1. Database

processing

  • 2. Peptide

analysis

  • 3. Subgroup

identification

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SLIDE 5

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

Database processing

  • 1. Database

processing

  • 3. Subgroup

identification

IMGT database In silico digestion MCs and PTMs FR coverage LC subgroup

  • 2. Peptide

analysis

New DB

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

Peptide analysis

  • 1. Database

processing

  • 3. Subgroup

identification

Peptide frequency in the subgroup Median FR coverage of the peptide Output: ASCII file containing the peptide mass, the frequency, the median FR coverage and the subgroup

  • 2. Peptide

analysis

m/z Freq. FR cov. Class 801 0.1 0.3 LC1 1100 0 0.8 LC10 1398 0.5 LC1 2100 0 LC3 2168 0.2 0.1 LC1 … … … …

ASCII file

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

Subgroup identification

Comparison between MS spectra and peptides in the ASCII file (tolerance window in Da or ppm) Subgroup identification

  • 1. Database

processing

  • 3. Subgroup

identification

  • 2. Peptide

analysis

Peak list

  

  

      

N n S m n S m N n S m n N n S m n

FR Int S Score FR Freq Int S Score Freq Int S Score

1 ) ( 3 ) ( 1 ) ( 2 1 ) ( 1

) ( ) ( ) (

ASCII file

m/z Int 801 25.0 1100 39.0 1400 15.0 1406 100.0 1512 24.0 … …

m/z Freq. FR cov. Class 801 0.1 0.3 LC1 1100 0 0.8 LC10 1398 0.5 LC1 2100 0 LC3 2168 0.2 0.1 LC1 … … … …

N matching peaks

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

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

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

Thanks!