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Screening of Neoantigen HLA Complexes | LifeTein Peptide Blog

Mutated peptides, known as neoantigens, derived from a patient’s cancer genome can be targeted by T-cell immunity. However, identifying which peptides can be presented by MHC molecules and stimulate T cells has proven challenging. Existing algorithms can predi

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This guide cannot diagnose a condition or recommend a personal treatment plan. Discuss medical questions with a qualified professional.

Mutated peptides, known as neoantigens, derived from a patient’s cancer genome can be targeted by T-cell immunity. However, identifying which peptides can be presented by MHC molecules and stimulate T cells has proven challenging. Existing algorithms can predict MHC binding but struggle to account for the half-lives of these complexes (a critical immunological parameter called kinetic stability). Enhancing our ability to determine the true stability of neoantigen peptide/MHC complexes is crucial, as only a small fraction of peptides in current vaccines effectively trigger CD8+ T-cell responses.

A study utilized a rapid, high-throughput approach to experimentally measure peptide/HLA thermal stability on a scale needed for analyzing neoantigens from thousands of patients. By combining UV-cleavable peptide/HLA class I complexes with differential scanning fluorimetry, the Tm values of neoantigen complexes were determined. These Tm values were accurate, reproducible, and directly proportional to the complexes’ half-lives. When analyzing known HLA-A2–restricted immunogenic peptides, Tm values correlated more strongly with immunogenicity than algorithm-predicted binding affinities. Using temperature stability information can help select neoantigens for cancer vaccines, focusing on mutated peptides most likely to be expressed on the cell surface.

Tm analysis of HLA-A2 complexes containing immunogenic neoantigen peptides

ME-1

FLDEFMEGV

63.2 ± 0.4

FNDC3B

VVMSWAPPV

61.6 ± 0.6

PRDX5

LLLDDLLVSI

55.3 ± 0.7

GAS7

SLADEAEVYL

59.2 ± 0.7

KIAA0223

VLHDDLLEA

59.7 ± 0.5

GAPDH

GIVEGLITTV

58.5 ± 0.6

HSP70

SLFEGIDIYT

59.8 ± 0.6

ACTININ

FIASNGVKLV

56.9 ± 0.5

HAUS3

ILNAMIAKI

56.9 ± 0.2

CSNK1A1

GLFGDIYLAI

52.6 ± 0.6

CLPP

ILDKVLVHL

57.1 ± 0.4

CDK4

ACDPHSGHFV

59.9 ± 0.3

AHNAK

FMPDFDLHL

60.1 ± 0.2

SRPX

TLWCSPIKV

63.7 ± 0.1

COL18A1

VLLGVKLFGV

60.9 ± 0.3

ERBB2

ALIHHNTYL

59.4 ± 0.5

TEAD1

VLENFTIFLV

48.2 ± 0.2

SVLENFTIFL

55.8 ± 0.2

NSDHL

ILTGLNYEV

61.4 ± 0.1

GANAB

ALYGFVPVL

61.7 ± 0.2

CDC37L1

FLSDHLYLV

61.8 ± 0.1

FLNA

HIAKSLFEV

56.4 ± 0.4

SPOP

FLLDEAIGL

60.7 ± 0.1

ACPP

VLAKKLKFV

56.8 ± 0.7

DCAKD

LLHTELERFL

42.9 ± 0.2

CIT

TLLSQVNKV

53.2 ± 0.3

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Protein-Protein Interaction Studies

In FRET-based assays, TAMRA acts as an acceptor dye paired with donors like fluorescein. This configuration allows detection of molecular interactions between labeled peptides and target proteins. For instance, TAMRA-labeled kinase substrate peptides can reveal enzymatic activity by quantifying changes in FRET efficiency upon phosphorylation. Find other fluorescent pairs here.

Source: lifetein.com ↗

Can I purchase custom FAM-labeled peptides for my research?

Yes. Specialized providers such as LifeTein offer custom synthesis of FAM-labeled peptides with high purity (>98%) and rigorous analytical validation. Their services include choices between 5-FAM and 6-FAM, optional spacers (Ahx, β-Ala), and a variety of conjugation positions (N-terminus, lysine side chain, cysteine-specific labeling).

Source: lifetein.com ↗
Practical and safety references

These excerpts are educational, not personalised medical instructions.

How-to reference

How to solubilize my synthetic peptides? #

Please refer to this FAQ for details: Handling and Storage of Synthetic Peptides. If the peptides are still cloudy, or turbid, you may have reached the limit of solubility. When the peptides are insoluble in the buffer, please try to sonicate, centrifuge, and lyophilize the peptide. Make sure to break the lyophilized lumps into a fine powder. Then try a small volume of a good agent 8M Urea, NMP, DMF, or DMSO to dissolve the peptide. Then dilute with water or your desired buffer. For peptides with Arg or LYs, you should try to lower the pH to 6 because the protonated amino acids will help solubility. Sonication and the following solvents may help with difficult peptides: 1) Begin with 100 % acetonitrile then dilute with water until 50% 2) Begin with 100% DMSO then dilute with water until 30 % 3) Dissolve it with 8M Urea 4) Dissolve it with 6 or 8 M Guanidine hydrochloride 5) 6M GuHCL, 0.05% TFA, pH2, 6) 100% TFA 7) 40% AcOH, 30%ACN, 30% water

Source: lifetein.com ↗
Storage reference

Storage temperatures and conditions #

For many of our antibodies, freezing at -20 C or -80 C in small aliquots is the optimal storage condition. Aliquotting minimizes damage due to freezing and thawing, as well as contamination introduced by pipetting from a single vial multiple times. Aliquots should be no smaller than 10 µl. Upon receiving the antibody, centrifuge at 5,000 x g for 30 seconds to pull down the solution, and transfer aliquots into low-protein-binding microcentrifuge tubes. Antibodies should be frozen as soon as possible, storage at 4 C upon receipt of the antibody is acceptable for one to two weeks, followed by freezing for long-term storage. To prevent microbial contamination, sodium azide can be added to an antibody preparation to a final concentration of 0.02% (w/v). If using antibodies for in vivo studies, please be sure to use preparations that do not contain sodium azide. This antimicrobial agent blocks the cytochrome electron transport system. Sodium azide will interfere with any conjugation that involves an amine group and should be removed before proceeding with the conjugation. After conjugation, antibodies can be stored in sodium azide but 0.01% thimerosal (Merthiolate), which does not have a primary amine, is an acceptable alternative. Sodium azide can be removed from antibody solutions by dialysis or gel filtration. The molecular weight of IgG is 150,000 daltons (IgM is ~ 600,000); the molecular weight of sodium azide is 65 daltons. A micro-dialysis unit with a cut off at 14,000 dalt…

Source: lifetein.com ↗
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