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- Analysis report


Date: 2021-09-22

Submitted by: Isabelle Sirois

Analysis type: Class I

Description of experiment:

MHC CLASS I MOUSE EL4 USER2 - 3 BIOLOGICAL REPLICATES

Samples:

MHC1_EL4_USER2_N1: Alleles: H-2-Kb, H-2-Db MHC1_EL4_USER2_N2: Alleles: H-2-Kb, H-2-Db MHC1_EL4_USER2_N3: Alleles: H-2-Kb, H-2-Db

Species: MOUSE - EL4

# of cells: 100 M

Lysis buffer: 0.5% CHAPS

Type of beads: 80 mg Sepharose CNBr-activated beads

Antibody: M1 pan H2

Incubation time: overnight

MHC-ligand complex elution buffer: 1% TFA

Peptide elution buffer: 28% ACN/0.1% TFA

Type of MS/MS: ORBITRAP ECLIPSE TRIBID MASS SPECTROMETER

Peptide identification software: PEAKS V10.6

Peptide FDR: 1%


Sample Overview

- LF Score: fraction of peptides between 8 and 12 mers. - BF Score: fraction of peptides between 8 and 12 mers which are predicted to be strong or weak binders.

Sample Total peptides Peptides between 8-12 mers LF Score BF Score
MHC1_EL4_USER2_N1 1723 1645 0.95 0.83
MHC1_EL4_USER2_N2 1767 1679 0.95 0.89
MHC1_EL4_USER2_N3 1846 1745 0.95 0.88
UpSet Plot (only displaying intersections containing >= 1% of at least one sample)

Peptide Length Distribution (maximum of 30 mers)


Annotation Results

NetMHCpan eluted ligand predictions made for all peptides between 8 & 12 mers, inclusive. - Percent rank cutoffs for strong and weak binders: 0.5 and 2.0. - Percentages are calculated across rows (i.e. percentage of total peptides for a respective sample).

Sample Total peptides Allele Strong binders Weak binders Non-binders

MHC1_EL4_USER2_N1

1645 H-2-Kb 743 (45.2%) 227 (13.8%) 675 (41.0%)
H-2-Db 603 (36.7%) 203 (12.3%) 839 (51.0%)

MHC1_EL4_USER2_N2

1679 H-2-Kb 936 (55.7%) 236 (14.1%) 507 (30.2%)
H-2-Db 508 (30.3%) 233 (13.9%) 938 (55.9%)

MHC1_EL4_USER2_N3

1745 H-2-Kb 981 (56.2%) 237 (13.6%) 527 (30.2%)
H-2-Db 534 (30.6%) 246 (14.1%) 965 (55.3%)
Binding Affinities

Binding Heatmaps

NetMHCpan eluted ligand predictions made for all peptides between 8 & 12 mers, inclusive. Approximate color legend (detailed mapping shown next to heatmaps):

Predicted strong binders (%rank <= 0.5)

Predicted weak binders (0.5 < %rank <= 2.0)

Predicted non-binders

- - # of peptides in sample


Sequence Motifs

Clustering performed with all peptides between 8 & 12 mers, inclusive.

- Percentages represent the percentage of peptides in a given group predicted to strongly bind the indicated allele.

Polar

Neutral

Basic

Acidic

Hydrophobic

MHC1_EL4_USER2_N1 (peptides clustered: 1645, outliers: 16)

Peptides in group: 202

H-2-Kb: 13%,

H-2-Db: 18%

Peptides in group: 448

H-2-Kb: 87%,

H-2-Db: 6%

Peptides in group: 114

H-2-Kb: 0%,

H-2-Db: 0%

Peptides in group: 330

H-2-Kb: 84%,

H-2-Db: 10%

Peptides in group: 535

H-2-Kb: 9%,

H-2-Db: 95%

MHC1_EL4_USER2_N2 (peptides clustered: 1679, outliers: 19)

Peptides in group: 782

H-2-Kb: 90%,

H-2-Db: 4%

Peptides in group: 431

H-2-Kb: 11%,

H-2-Db: 94%

Peptides in group: 221

H-2-Kb: 67%,

H-2-Db: 11%

Peptides in group: 226

H-2-Kb: 16%,

H-2-Db: 19%

MHC1_EL4_USER2_N3 (peptides clustered: 1745, outliers: 46)

Peptides in group: 248

H-2-Kb: 13%,

H-2-Db: 16%

Peptides in group: 986

H-2-Kb: 90%,

H-2-Db: 7%

Peptides in group: 465

H-2-Kb: 12%,

H-2-Db: 92%
MHC1_EL4_USER2_N1 sequence motif(s)
H-2-Kb

Peptides: 964

H-2-Db

Peptides: 796

Non-binders group 2

Peptides: 126

Non-binders group 1

Peptides: 128

MHC1_EL4_USER2_N2 sequence motif(s)
H-2-Kb

Peptides: 1161

H-2-Db

Peptides: 736

Non-binders group 2

Peptides: 65

Non-binders group 1

Peptides: 114

MHC1_EL4_USER2_N3 sequence motif(s)
H-2-Kb

Peptides: 1208

H-2-Db

Peptides: 777

Non-binders group 2

Peptides: 134

Non-binders group 1

Peptides: 57