Ihe Thermo Fisher's New Immunopeptidomics Kit Pụtara maka Omenala Peptide Workflows
Odida obodo nke nyocha ihe ngosi antigen na nchọpụta neoantigen na-enwe nnukwu mgbanwe teknụzụ. Azụmahịa nke nkwadebe nlele ahaziri ahazi yana ngwa ngụta ezubere iche—nke kachasị Thermo Fisher Scientific's SureQuant IP-MS ngwa nkwadebe sample na Pierce MHC Class I Sistemu kewapụ — ewetala mmụgharị na-enwetụbeghị ụdị ya na immunopeptidomics dabere na spectrometry..
N'akụkọ ihe mere eme, Immunopeptidomics dabere na nnweta data dabere na enweghị ebumnuche (DDA) ịchọpụta peptides nke MHC/HLA sitere na lysates cell dị mgbagwoju anya. Ebe ọ dị irè maka nchọpụta ebumnuche mbụ, Usoro ọrụ DDA na-enweghị atụ nwere ahụhụ site na mgbanwe dị elu na-agba ọsọ na-agba ọsọ, mmetụta dị ala maka antigens dị ala, na enweghị atụ n'etiti ụlọ nyocha.
Na ngwa ngwa ahaziri ahazi, ụlọ ọrụ ahụ na-aga n'ihu n'ike n'ike gaa n'ọnụ ọgụgụ ezubere iche—kpọmkwem Nleba anya Ọkọlọtọ Parallel Reaction nke Internal. (IS-PRM) na SureQuant ezubere iche usoro ọrụ. Usoro evolushọn a na-emegharị ozugbo ihe achọrọ etinyere na peptides synthetic omenala elu. peptides ntụaka sịntetik na akara isotoppe kwụsiri ike (SIL) ụkpụrụ ime abụghịzi naanị njikwa njikwa nhọrọ; ha bụ integral quantitative reagents na-achịkwa assay sensitivity, ike nso, na uka spectrometric ziri ezi.
Ngbanwe site na nchọpụta gaa na ọnụọgụ nke ezubere iche na Immunopeptidomics
Ngwa ndị ahaziri ahazi na-akwalite nkwalite immunoaffinity site na iji bead ndọta nwere njikọ agbakwunyere akwadoro., kachasị lysis buffers, yana ụkpụrụ mgbari microfluidic akpaghị aka. Agbanyeghị, ezi ike nyocha nke ngwa ndị a na-emeghe ma ọ bụrụ na ejikọtara ya na spectrometry mass spectrometry ezubere iche na oke nleba anya Orbitrap mass spectrometers..
Mweghara mgbochi ọrịa <– MHC-I/II Akpụkpọ anụ ejikọtara ọnụ na-akwadobu SIL Spike-Na ọkọlọtọ <- Peptides sịntetik dị arọ nke omenala (Ọnụ ego AAA)
Ọkọlọtọ Immunopetidomics Workflow Cell Lysate / Tissue v v Acid Elution & Hichapụ <- Nkewa nke HLA Chains dị arọ & Peptides v v Ezubere iche nanoLC-MS/MS <- SureQuant IS-PRM / Orbitrap dị elu
N'ime nyocha IS-PRM ezubere iche, sịntetik arọ isotope akpọrọ peptides dabara na neoantigens a na-enyo enyo ma ọ bụ antigens metụtara tumor (Ọ bụ ya) na-siked n'ime sample tupu LC-MS analysis. Mass spectrometer na-enyocha mgbanwe ion nkenke maka ma peptide “ọkụ” endogenous. (m/z ọkụ) na ọkọlọtọ ime "dị arọ" sịntetik (m/z arọ).
Dị ka e gosiri na nso nso a akwụkwọ, dị ka a Ọmụmụ PubMed na nleba anya HLA peptide quantification site IS-PRM, Uzo a ezubere iche na-enweta uche n'ọkwa atomole, na-eme ka nkwenye obi ike nke ọnụọgụ MHC dị ala nke a ga-atụfu na nyocha na-enweghị atụ..
Ntugharị igodo: Nkwenye nke ngwa ihe nleba anya nke immunopeptidomics ọkọlọtọ na-akpali ubi ahụ site na njirimara qualitative gaa na nkwenye ọnụọgụ siri ike.. N'ihi ya, peptides ntụaka sịntetik ga-ezute kemịkalụ siri ike, isotopic, yana nkọwa anụ ahụ iji zere imebi nyocha LC-MS ezubere iche.
Nkọwapụta Peptide dị elu: Kedu ihe ngwa ngwa chọrọ
Mgbe ndị nchọpụta na-ejikọta ngwa immunopeptidomics ezubere iche n'ime pipeline ha, Ekwesịrị ịmegharị nkọwapụta peptide omenala n'elu. Ọkọlọtọ agụmakwụkwọ-ọkwa sịntetik peptides (eg., 70-80% ịdị ọcha na-adị ọcha na ihe mgbochi) ewebata arịa nyocha dị mkpa n'ime sistemụ nanoLC-MS/MS.
- Mmetụta ion-Pairing: TFA na-ebute mbelata mgbaàmà ESI siri ike na nanoLC-MS
- Ọdịmma Gravimetric: Mmiri + counterions ewebata 10-30% njehie arọ
- Ngwọta: 6M HCl microwave hydrolysis + LC-MS AAA maka net peptide ọdịnaya
Ogidi Ọdịmma Peptide Omenala Dị Mkpa Glp 1 Ụlọ ọrụ Tripeptide
- Chemical & Isotopic ịdị ọcha
- RP-HPLC ịdị ọcha: >95% ka >98%
- Ịba ụba Isotopic: ≥99 atọm % 13C na 15N (C-terminal Lys/Arg)
- Trifluoroacetate (TFA) Mwepụ mgbochi
- Nkọwapụta: Ngbanwe nnu na acetate/HCl Nke Peptides (Mfọdụ TFA < 1.0%)
- Ọnụ ọgụgụ zuru oke site na nyocha Amino Acid (AAA)
Ịba ụba Isotopic na ọnụ ụzọ ịdị ọcha nke Isotopic
Maka nyocha IS-PRM ezubere iche na SureQuant, kwụsiri ike isotope-akara (SIL) A na-ahazi ụkpụrụ ime site na itinye amino acid dị arọ na C-terminus, na-emekarị [^{13}C₆, ^{15}N₂]Lysine (Δ m = +8.0142 Na) ma ọ bụ [^{13}C₆, ^{15}N₄]Arginine (Δ m = +10.0083 Na).
Iji kwe nkwa nzizi nha anya zuru oke:
- Isotopic ịdị ọcha: Ihe mmalite amino acid dị arọ ga-enwerịrị ≥ 99 atọm % a ^{13}C na ^{15}N nkwalite.
- Ọnụego mmetọ enweghị akara: Mmetọ peptide “ọkụ” enweghị akara n'ime ọkọlọtọ dị arọ ga-abụrịrị < 0.1%. Ọkwa dị elu nke adịghị ọcha nke ọkụ na-eji aka aka wulite nha peptide endogenous.
- Iguzosi ike n'ezi ihe nke Mass Shift: Nke +8 Da ma ọ bụ +10 Da mass shift na-eme ka o doo anya na ọdụ ụgbọ mmiri isotopic site na ọkọlọtọ dị arọ anaghị ejikọta na windo monoisotopic m/z nke endogenous analyte..
Mwepụ mgbochi: Mbelata Mbelata ion TFA
N'oge siri ike-phase Peptide Synthesis (SPSS), ikpochapu site na resin na nke na-esote Reverse-Phase HPLC (RP-HPLC) ịdị ọcha na-adabere kpamkpam na trifluoroacetic aci
Electrospray ionization (ESI) Mgbochi: CF_3COO^- ion na-etolite ụzọ abụọ ion gas siri ike nwere nnukwu ụgwọ isi H36 Peptide N'ogbe foduru (Lys^+, Arg^+, na N-terminal NH_3^+), na-ewepụ ụgwọ peptide na-ebelata ma na-ebelata arụmọrụ ionization nke ukwuu.
ọng>: CF_3COO^- ion na-etolite ụzọ abụọ ion gas siri ike nwere ihe ndị fọdụrụ na-akwụ ụgwọ nke ọma (Lys^+, Arg^+, na N-terminal NH_3^+), na-ewepụ ụgwọ peptide na-ebelata ma na-ebelata arụmọrụ ionization nke ukwuu.
- Ihe Njide Chromatographic: TFA fọdụrụnụ na-arụ ọrụ dị ka ion-pairing reagent siri ike, na-agbanwe oge nanoflow mmiri chromatography na-eme ka ọdụdụ dị elu.
⚠️ Ịdọ aka ná ntị: Iji peptides ntụaka sịntetik nwere TFA fọdụrụnụ na nanoLC-MS/MS nwere ike ịkwụsị ionization nke mkpokọta MHC peptides dị ala na-abawanye ruo 80%, na-eduga na adịghị mma ụgha na nkwado neoantigen.
Immunopeptidomics arụ ọrụ na-ahazi na-enye iwu mgbanwe mgbanwe. Site n'ịgbanwe nnu peptide site na trifluoroacetate ka ọ bụrụ acetate (CH_3COO^-) ma ọ bụ hydrochloride (Cl^-), A na-ebelata ọdịnaya TFA fọdụrụnụ ka < 1.0%, na-eme ka nzaghachi mgbaàmà nanoESI kwụsiri ike.
Nkwenye ntinye uche zuru oke site na nyocha Amino Acid (AAA)
Na nyocha SureQuant ezubere iche, izi ezi nke ọnụọgụ peptide endogenous dabara na izi ezi nke mkpokọta ọkọlọtọ SIL dị n'ime..
Na-adabere na nha gravimetric mass (na-atụ lyophilized peptide ntụ ntụ) bụ ihe ebumpụta ụwa ezighi ezi n'ihi mgbanwe hydration (5-15 wt%) na mpụta counterion uka (10-25 wt%). Otu vial tụrụ ka 1.0 mg nke peptide ntụ ntụ nwere ike ịnwe naanị 0.70 mg nke peptide net arụ ọrụ.
Iji nweta nhazi ihu igwe:
- Microwave Acid Hydrolysis: A na-etinye peptide hydrolyzed n'ime 6 M HCl na 110°C ma ọ bụ n'okpuru igwe ọkụ ọkụ maka awa 1-2.
- Chromatography Liquid-Mass Spectrometry (LC-MS) AAA: A na-agụta amino acid n'efu na mmiri megide ụkpụrụ ntụaka NIST nwere ike ịchọpụta, dị ka akọwara na a Journal of Proteome Research analysis na amino acid analysis maka zuru ezu peptide quntitation.
- Ọdịnaya Peptide Net (NPC): A na-edekọ kpọmkwem molarity na pasent NPC na Asambodo nyocha (CoA), na-eme ka nhazi ọkwa ọkwa picomole ziri ezi.
Ntugharị Dynamics na chọrọ nhazi usoro dị elu
Ntụgharị asụsụ ụlọ ọgwụ nke immunopeptidomics-dị ka mmepe ọgwụ mgbochi ọrịa kansa ahaziri iche yana nkwado antigen ọgwụgwọ cell—na-amanye usoro iheomume siri ike.. Ndị na-eme nchọpụta na-eji usoro nhazi ihe nlele akpaaka enweghị ike ị nweta oge ntụgharị ọtụtụ ọnwa maka peptides omenala.
Nnyefe Immunopeptidomics dị elu nke Matrix Neoantigen Target Prediction → Na Silico HLA Binding Algorithms Ngwa Ngwa Ngwa Ngwa Ngwa → 10 - 14 Ntugharị ụbọchị azụmahịa → 96-Well Micro-Aliquoted Format Vessel Surface Optimization → Polypropylene dị ala / LC-MS/MS Assay eburu ụzọ gbasasịa → Nkwado Attomole site na SureQuant IS-PRM
Isi mmegharị arụ ọrụ akọwara na a Protocol PMC na nkwadebe ihe nlele immunopeptidomics gụnyere:
- 10– 14 Day ngwa ngwa-Turnaround Synthesis: Ngwa nyiwe SPPS akpaghị aka nwere ike ịmekọrịta 96 ka 384 peptides dị iche iche n'otu oge.
- 96-Ọfọn Pre
Mbelata mgbasa ozi na-abụghị nke akọwapụtara: peptides na-ejikọta HLA na-abụkarị hydrophobic 8-11-mers (Klas I) ma ọ bụ 12-25-mrs (Klas II) nke na-arapara ngwa ngwa na elu plastik ọkọlọtọ. Micro-aliquoting n'ime arịa polypropylene dị ala ma ọ bụ na-ebunye peptides na matrices DMSO/ACN agbazere na-egbochi mfu ihe atụ hydrophobic.. Peptide Synthesis
d plastic elu. Micro-aliquoting n'ime arịa polypropylene dị ala ma ọ bụ na-ebunye peptides na matrices DMSO/ACN agbazere na-egbochi mfu ihe atụ hydrophobic..
Mmegharị bara uru maka ndị na-enye Peptide omenala
Iji nyere ndị nyocha aka iji obi ike were ngwa ngwa immunopeptidomics ọkọlọtọ, Ndị na-eweta peptide synthesis ga-esi na ụlọ ahịa mmezu nke na-agafe agafe ghọọ ndị mmekọ ihe ngwọta pụrụ iche..
Ndozi usoro maka mmeghari ndị na-enye Peptide omenala 1: Immunopeptidomics echekwabara-katalọgụ dị njikere & Ụlọ akwụkwọ arịlịka
- Ogwe ntụnye aka emebere nke ọma maka alleles nkịtị (HLA-A02:01, A24:02) Ntughari 2: Akụkụ QC ezubere iche & Asambodo nyocha nke ọtụtụ metric
- ESI-HRMS nha nha nha (< 2 ppm), RP-HPLC akara, ihe fọdụrụ TFA %, na mgbanwe AAA 3: Ịkpọ aha ngwa ngwa & Ọrụ mgbanwe pụrụ iche
- Peptide Synthesis Mgbakwunye isotope kwụsiri ike, Mkpado N/C-ọnụ, na Klas 100 sterile QC
Immunopeptidomics echekwabara-katalọgụ dị njikere & Akụkụ Allele akọwapụtara
Ndị na-enye peptide kwesịrị inye curated, Ogwe ntụaka ndị na-anọghị na shelf dabere na njirimara arịlịka nke HLA allele mara mma..
- Ogwe Klas I: Na-ekpuchi alleles dị elu dị ka HLA-A02:01, HLA-A24:02, HLA-B07:02, na HLA-C07:02. Ogwe ndị a nwere onye anaghị akpọ aha kwadoro ($9$-mer) na decamer ($10$-mer) peptides na canonical arịlịka fọdụrụnụ (eg., Leu/ zutere na ọnọdụ 2 na Val/L-Leu na ọnọdụ 9 maka HLA-A*02:01).
- Klas II panel: Mkpuchi HLA-DRB101:01, HLA-DRB104:01, na HLA-DQ8, na-etinye ogologo usoro 13–17-mer nwere njirimara solubility enwetara.
Inwe oghere ndị na-anọghị na shelf dị maka izipu acc ozugbo
Ndị na-enye ihe kwesịrị ịnye CoAs zuru oke nwere: Peptide Therapeutic Supplier
na njigide oge nhazi (eg., Mgbanwe nke ọnụahịa iRT) maka LC-MS na-agba.
Akụkụ QC ezubere iche na asambodo nyocha zuru oke
Njikwa Ogo siri ike (QC) panel maka immunopeptidomics ntụaka peptides ga-agbatịrịrị ihe nrịbama MALDI-TOF..
Ndị na-enye ihe kwesịrị ịnye CoAs zuru oke nwere:
- Isi Mkpebi ESI-HRMS Spectra: Na-enyocha steeti monoisotopic mass na ụgwọ ([M+H]^+, [M+2H]^{2+}, [M+3H]^{3+}) na nnukwu njehie < 2 ppm.
- Chromatogram RP-HPLC: Na-egosi >95% ma ọ bụ >98% ịdị ọcha kemịkalụ nwere ọnọdụ gradient doro anya.
- Mkpebi TFA fọdụrụnụ: Ion Chromatography (IC) ma ọ bụ ọnụọgụ 19F-NMR na-egosi TFA fọdụrụnụ < 1.0%.
- Ọdịnaya Peptide Net site na AAA: Bara uru nanomoles/vial ziri ezi iji kwe nkwa mwegharị n'ime ọkọlọtọ spike-ins.
Maka ndụmọdụ: Mgbe ị na-arịọ SIL peptides omenala maka immunopeptidomics ezubere iche, Gbaa mbọ hụ na onye na-ere ahịa na-enye ọdịnaya peptide net enwetara AAA yana mgbanwe nnu na-enweghị TFA. Otu nzọụkwụ a na-ewepụ isi mmalite nke nhụsianya n'etiti batch na nyocha Orbitrap LC-MS..
Labeling Arọ-Isotope Ọsọ na Ọrụ Micro-Aliquoting
Ndị na-enye ihe ga-emeziwanye njikọ nke peptides gbagwojuru anya, gụnyere:
- Incorporation nke Heavy Isotope Inc: High-arụmọrụ njikọ nke [^{13}C₆, ^{15}N₂]Lys na [^{13}C₆, ^{15}N₄]Arg na-enweghị isotopic dilution n'oge cleavage.
- Ọnụ & Mgbanwe nke ime: Biotinylation, njikọ nke fluorophore (eg., FITC, Cy5), N-terminal acetylation, na pịa-chemistry aka (azide/alkyne) maka multi-modal antigen nsuso.
- Nhazi ime ụlọ dị ọcha: Maka bioassays arụ ọrụ mgbada ala (eg., Nnwale ịgbalite cell T-cell, ELISpot, ma ọ bụ TCR njide nyocha), A ghaghị ịhazi peptides n'okpuru ọnọdụ ime ụlọ na-adịghị ọcha site na iji nyocha endotoxin (< 0.01 EU/µg).
Mmekọrịta Strategic: Na-ebuli usoro ọrụ Immunopeptidomics
Ịgagharị na mgbagwoju anya nke immunopeptidomics ezubere iche chọrọ nhazi nso n'etiti ndị na-emepụta ngwa reagent., akụrụngwa spectrometry bụ isi, na ndị mmekọ njikọ peptide pụrụ iche.
Dị ka onye ndu raara onwe ya nye n'ichepụta peptide, nke MOL na-agbanwe ikpo okwu njikọ peptide omenala na-enye akụrụngwa agbakwunyere ahaziri nke ọma iji kwado mmemme immunopeptidomics dị elu na mmemme nchọpụta neoantigen.
MOL na-agbanwe Injin Immunopeptidomics
SPPS Salt Exchange Platform AAA Quantitation Suite Class nwere nnukwu ihe 100 Ime ụlọ ọcha
→ Synthesis nke mgbagwoju anya & Hydrophobic Peptides → Mwepụ Mgbochi TFA (< 1.0% Mfọdụ TFA) → Ọdịnaya Peptide Net akwadoro & Kpọmkwem Molarity → Endotoxin-efu & Lyophilization Sterile
Site n'iji ike mgbanwe peptide dị elu yana ọrụ iwepụ TFA, Ndị na-eme nchọpụta nwere ike mechie ọdịiche dị n'etiti nkwadebe ihe nlele dabere na kit yana ntụkwasị obi dị elu ezubere iche maka nha LC-MS.. Ọzọkwa, mmepụta n'ime sterile klaasị 100 nrụpụta ụlọ dị ọcha ụlọ ọrụ na-ahụ na ụkpụrụ peptide sịntetik dabara adaba ozugbo maka ma nyocha nke oke spectrometry yana nkwado immunological dabere na cell..
Ngwa ihe nlere ahazi nke ọma agbanweela nhazi sample immunopeptidomics. Site na ijikọ ngwa ndị a na peptides synthetic omenala emebere maka ịdị ọcha isotopic siri ike, Mwepụ TFA, yana ọnụọgụ AAA kwadoro, ndị na-eme nchọpụta nwere ike ịmepụta ihe siri ike, pipeline dị elu nke na-eme ka nchọpụta neoantigen na immunotherapy na-aga n'ihu na ntụkwasị obi zuru oke.
