Seo Thermo Fisher's New Immunopeptidomics Kit e se Bolelang bakeng sa Ts'ebetso e Tloaelehileng ea Peptide Workflows
Sebopeho sa lipatlisiso tsa tlhahiso ea antigen le ho sibolloa ha neoantigen se ntse se fetoha phetoho e kholo ea theknoloji.. Ho rekisoa ha lisampole tse emeng le lisebelisoa tse reriloeng tsa palo-haholo-holo Thermo Fisher Scientific's SureQuant IP-MS lisebelisoa tsa ho lokisa sampole le Pierce MHC Class Isolation systems-e tlisitse ho ikatisa ho neng ho e-s'o ka ha e-ba teng pele ho immunopeptidomics e thehiloeng ho spectrometry..
Ho latela nalane, immunopeptidomics e ne e itšetlehile ka Phano e sa Rereloang ea Data-Dependent (DDA) ho sibolla li-peptide tse hlahisitsoeng ke MHC/HLA ho tsoa ho li-cell lysate tse rarahaneng. Ha e ntse e sebetsa molemong oa ho sibolla sepheo sa pele, Ts'ebetso ea DDA e sa lebelloang e bile le phapang e phahameng ea ho matha-to-run, kutlo e tlase bakeng sa li-antigen tse ngata haholo, le papiso e fokolang ea li laboratori.
Ka lisebelisoa tse tloaelehileng, indasteri e fetoha ka potlako ho ea ho palo e lebisitsoeng-haholo-holo Internal Standard Parallel Reaction Monitoring (KE-PRM) le SureQuant targeted workflows. Phetoho ena ea mekhoa e fetola ka kotloloho litlhoko tse behiloeng holim'a li-peptide tsa maiketsetso tse ka holimo. Li-peptide tsa maiketsetso tsa maiketsetso le li-isotope tse tsitsitseng tse ngotsoeng (SILE) maemo a ka hare ha e sa le taolo ea boleng ba boikhethelo feela; ke li-integral quantitative reagents tse laolang kutloisiso ea assay, dynamic range, le ho nepahala ha spectrometric ka bongata.
The Shift from Discovery to Targeted Quantitation in Immunopeptidomics
Li-kits tse tloahelehileng li ntlafatsa matla a ho itšireletsa mafung ka ho sebelisa lifaha tsa makenete tse kopaneng le li-antibody tse kopaneng., optimized lysis buffers, le li-protocol tsa automated microfluidic digestion. Leha ho le joalo, matla a 'nete a tlhahlobo ea lisebelisoa tsena a notlolloa ha a pentiloe ka "mass spectrometry" a phahameng-resolution Orbitrap mass spectrometers..
Tšoaetso ea Immunoaffinity <- Lifaha tse netefalitsoeng esale pele tsa MHC-I/II Antibody-Coupled SIL Spike-In Standard <- Li-peptide tse boima tse entsoeng ka maiketsetso (AAA Quantified)
E tloaelehileng ea Immunopeptidomics Workflow Cell Lysate / Tissue v v Acid Elution & Hloekisa <- Karohano ea liketane tse boima tsa HLA & Peptides v v Targeted nanoLC-MS/MS <- SureQuant IS-PRM / Orbitrap e phahameng-Resolution
Litlhahlobong tse lebisitsoeng tsa IS-PRM, li-peptide tse boima tse entsoeng ke isotope tse tšoanang le li-neoantigens tse belaelloang kapa li-antigen tse amanang le tumor. (Ho felile ke lehlohonolo) li kenngoa ka har'a sampole pele ho tlhahlobo ea LC-MS. Mass spectrometer e lekola liphetoho tse ikhethileng tsa ion bakeng sa "peptide" e bobebe. (m/z khanya) le maemo a ka hare a maiketsetso a "boima". (m/z boima).
Joalokaha ho bontšitsoe libukeng tsa morao-rao, joalo ka a Thuto ea PubMed mabapi le palo e lebisitsoeng ea HLA peptide ke IS-PRM, mokhoa ona o lebisitsoeng o finyella kutloisiso ea boemo ba attomole, e nolofalletsang netefatso e tiileng ea li-ligand tsa MHC tsa palo e tlase tse neng li ka hloloheloa tlhahlobo e sa reroang..
Key Takeaway: Kamohelo ea lisebelisoa tse emeng tsa immunopeptidomics prep kits e tsamaisa tšimo ho tloha ho boitsebahatso ba boleng ho isa ho netefatso e matla ea palo.. Ka lebaka leo, li-peptide tsa maiketsetso li tlameha ho kopana le lik'hemik'hale tse thata, isotopic, le litlhaloso tsa 'mele ho qoba ho sekisetsa litlhahlobo tse lebisitsoeng tsa LC-MS.
Litlhaloso tse phahameng tsa Peptide: Seo Standardized Kits Batla
Ha bafuputsi ba kopanya lisebelisoa tsa immunopeptidomics tse lebisitsoeng ho lipeipi tsa bona, Litlhaloso tsa peptide tse holimo li tlameha ho nchafatsoa. Li-peptide tsa maiketsetso tsa boemo ba thuto tse tloaelehileng (mohlala, 70-80% bohloeki bo litšila bo nang le li-counterion tse setseng) hlahisa li-artifact tsa bohlokoa tsa tlhahlobo ho nanoLC-MS/MS system.
- Phello ea Ion-Pairing: TFA e baka khatello e matla ea ESI ho nanoLC-MS
- Bothata ba Gravimetric: Mongobo + counterions hlahisa 10-30% phoso ea boima
- Tharollo: 6M HCl microwave hydrolysis + LC-MS AAA bakeng sa litaba tsa net peptide
Litšiea tse Hloahloa tsa Boleng ba Peptide Glp 1 Khamphani ea Tripeptide
- Lik'hemik'hale & Bohloeki ba Isotopic
- RP-HPLC Bohloeki: >95% ho >98%
- Matlafatso ea Isotopic: ≥99 athomo % 13C le 15N (C-terminal Lys/Arg)
- Trifluoroacetate (TFA) Ho tlosoa ha Counterion
- Tlhaloso: Phapanyetsano ea letsoai ho Acetate/HCl Ka li-peptides (TFA e setseng < 1.0%)
- Bongata bo felletseng ka Analysis ea Amino Acid (AAA)
Matlafatso ea Isotopic le Isotopic Purity Thresholds
Bakeng sa litlhahlobo tse lebisitsoeng tsa IS-PRM le SureQuant, e tsitsitseng ea isotope e ngotsoeng (SILE) litekanyetso tsa ka hare li entsoe ka ho kenyelletsa li-amino acid tse boima ho C-terminus, ka tlwaelo [^{13}C₆, ^{15}N₂]Lysine (Δ m = +8.0142 Le) kapa [^{13}C₆, ^{15}N₄]Arginine (Δ m = +10.0083 Le).
Ho netefatsa ho nepahala ha palo ea lipalo:
- Bohloeki ba Isotopic: Li-precursor tsa amino acid tse boima li tlameha ho ba le ≥ 99 athomo % a ^{13}C le ^{15}N ruisa.
- Sekhahla sa Tšilafalo e sa Ngoloang: Tšilafalo ea "peptide" e sa ngolisoeng ka har'a maemo a boima e tlameha ho ba < 0.1%. Boemo bo phahameng ba ho se hloeke ha leseli bo kenya litekanyo tsa endogenous peptide.
- Mass Shift Botšepehi: The +8 Da kapa +10 Da mass shift e netefatsa hore enfelopo ea isotopic e tsoang maemong a boima ha e kopane le fensetere ea monoisotopic m/z ea endogenous analyte..
Ho tlosoa ha Counterion: Ho fokotsa khatello ea TFA Ion
Nakong ea Solid-Phase Peptide Synthesis (SPSS), cleavage ho tloha ho resin le ho latela Reverse-Phase HPLC (RP-HPLC) tlhoekiso e itšetlehile haholo ka trifluoroacetic acid
Electrospray Ionization (ESI) Khatello: CF_3COO^- li-ion li theha li-ion tsa khase tse matla tse nang le motheo o nang le chaji e ntle H36 Peptide Wholesale masalla (Lys^+, Arg^+, le N-terminal NH_3^+), ho fokotsa litefiso tsa peptide le ho fokotsa ts'ebetso ea ionization haholo.
ong>: CF_3COO^- li-ion li theha li-ion tsa khase tse matla tse nang le masalla a mantlha a nang le tjhaji e ntle (Lys^+, Arg^+, le N-terminal NH_3^+), ho fokotsa litefiso tsa peptide le ho fokotsa ts'ebetso ea ionization haholo.
- Chromatographic Retention Drift: TFA e setseng e sebetsa e le setsi se matla sa hydrophobic ion-pairing, ho fetola linako tsa ho boloka nanoflow liquid chromatography le ho baka mohatla o phahameng.
⚠️ Tlhokomeliso: Ho sebelisa li-peptide tsa maiketsetso tse nang le masala a TFA ho nanoLC-MS/MS ho ka hatella ionization ea li-peptide tse ngata tse tlase tsa MHC ho fihlela ho. 80%, ho lebisa ho tse mpe tse fosahetseng netefatsong ea neoantigen.
Mokhoa o hlophisitsoeng oa immunopeptidomics workflows o laela ts'ebetso ea phapanyetsano ea counterion. Ka ho fetola letsoai la peptide ho tloha trifluoroacetate ho ea acetate (CH_3COO^-) kapa hydrochloride (Cl^-), mesaletsa ea TFA dikahare fokotswa ho < 1.0%, ho netefatsa karabo e tsitsitseng ea nanoESI.
Netefatso e Felletseng ea Concentration ka Tlhahlobo ea Amino Acid (AAA)
Litlhahlobong tse lebisitsoeng tsa SureQuant, ho nepahala ha palo ea endogenous peptide quantification e lekana ka kotloloho le ho nepahala ha spiked SIL internal concentration concentrations.
Ho itšetlehile ka tekanyo ea boima ba gravimetric (boima ba lyophilized peptide phofo) ha e nepahale ka tlhaho ka lebaka la ho feto-fetoha ha metsi (5-15 wt%) le masalla counterion boima (10-25 wt%). Vial e ne e le boima ba 1.0 mg ea phofo ea peptide e ka ba le feela 0.70 mg ea peptide e sebetsang ea net.
Ho fihlella tekanyo e nepahetseng ea molar:
- Microwave Acid Hydrolysis: Li-peptide li entsoe ka hydrolyzed 6 M HCl ka 110°C kapa ka tlas'a microwave e futhumatsang lihora tse 1-2.
- Mokelikeli Chromatography-Mass Spectrometry (LC-MS) AAA: Li-amino acid tsa mahala tsa Hydrolyzed li lekantsoe khahlano le litekanyetso tse ka lateloang tsa NIST, joalokaha ho hlalositsoe ho a Journal of Proteome Research analysis on amino acid analysis for absolute peptide quantitation.
- Net Peptide Content (NPC): Molarity e nepahetseng le peresente ea NPC li tlalehiloe Setifikeiting sa Tlhahlobo (CoA), e nolofalletsang litokiso tse nepahetseng tsa boemo ba picomole.
Turnaround Dynamics le Litlhoko tsa Sebopeho sa Boemo bo Phahameng
Phetolelo ea bongaka ea immunopeptidomics - joalo ka nts'etsopele ea ente ea mofets'e ea motho le netefatso ea antigen ea cell therapy - e beha linako tsa ts'ebetso tse thata.. Bafuputsi ba sebelisang li-platform tsa ho itokisa tsa mehlala ha ba khone ho fana ka nako ea likhoeli tse ngata bakeng sa li-peptide tse tloaelehileng.
High-Throughput Immunopeptidomics Delivery Matrix Neoantigen Target Prediction → Ka Silico HLA Binding Algorithms Rapid Custom Synthesis → 10 - 14 Phetoho ea Matsatsi a Khoebo Phetoho e Tšoanang ea Phetoho → Ntlafatso ea Sebōpeho sa Sejana sa 96-Well Micro-Ali qotsitsoeng → Polypropylene e Tlamang Tlase / LC-MS/MS E Lebisitsoeng Pele E Hlahiloe → Tlhahlobo ea Attomole ka SureQuant IS-PRM
Liphetoho tsa bohlokoa tsa ts'ebetso tse hlalositsoeng ho a Protocol ea PMC mabapi le tokiso ea sampole ea immunopeptidomics kenyeletsa:
- 10-Matsatsi a 14 a ho Fetola ka Potlako: Li-platform tsa SPPS tse tsamaisanang tse ikemetseng tse khonang ho kopanya 96 ho 384 li-peptide tse ikhethang ka nako e le 'ngoe.
- 96-Hantle Pele
Non-Specific Adsorption Mitigation: Li-peptide tse nang le HLA li na le li-hydrophobic 8-11-mers (Sehlopha sa I) kapa 12-25-mers (Sehlopha sa II) tse khomarelang habonolo holim'a polasetiki e tloaelehileng. Micro-aliquoting ka har'a likepe tse tlamang tse tlase tsa polypropylene kapa ho fana ka li-peptide ka har'a matrices a DMSO/ACN a qhibilihang pele ho thibela tahlehelo ea sampole ea hydrophobic.. Peptide Synthesis
d bokaholimo ba polasetiki. Micro-aliquoting ka har'a likepe tse tlamang tse tlase tsa polypropylene kapa ho fana ka li-peptide ka har'a matrices a DMSO/ACN a qhibilihang pele ho thibela tahlehelo ea sampole ea hydrophobic..
Liphetoho tse sebetsang bakeng sa Bafani ba Peptide ba Tloaelehileng
Ho thusa bafuputsi ho amohela lisebelisoa tse emeng tsa immunopeptidomics ka kholiseho, Bafani ba peptide synthesis ba tlameha ho tloha mabenkeleng a phethahatsang taelo ho ea ho balekane ba khethehileng ba tharollo..
Liphetoho tsa Leano bakeng sa ho Ikamahanya le Bafani ba Peptide ba Tloaelehileng 1: Li-Catalogs tse Lokisitsoeng tsa Immunopeptidomics & Lilaebrari tsa Anchor
- Liphanele tsa litšupiso tse entsoeng esale pele bakeng sa alleles tse tloaelehileng (HLA-A02:01, A24:02) Ho ikamahanya le maemo 2: Liphanele tsa QC tse lebisitsoeng & Li-Certificate tsa Multi-Metric tsa Tlhahlobo
- ESI-HRMS ho nepahala ka bongata (< 2 ppm), RP-HPLC mesaletsa, TFA e setseng %, le AAA Adaptation 3: Rapid Labeling & Litšebeletso tse khethehileng tsa ho fetola
- Peptide Synthesis Kenyelletso e tsitsitseng ea isotope, Li-tag tsa N/C-terminal, le Sehlopha 100 QC e hloekisitsoeng
Li-Catalogs tse Lokisitsoeng tsa Immunopeptidomics & Allele-Specific Panels
Bafani ba li-peptide ba lokela ho fana ka li-curated, liphanele tsa litšupiso tse kantle ho sethala tse ipapisitseng le li-HLA allele anchor motifs tse tsebahalang.
- Liphanele tsa Sehlopha sa I: Ho koahela li-allele tse phahameng-frequency joalo ka HLA-A02:01, HLA-A24:02, HLA-B07:02, le HLA-C07:02. Liphanele tsena li na le li-nonamer tse netefalitsoeng ($9$-mer) le decamer ($10$-mer) li-peptide tse nang le masalla a ankora a canonical (mohlala, Leu/Met at Position 2 le Val/L-Leu Maemong 9 bakeng sa HLA-A*02:01).
- Liphanele tsa Sehlopha sa II: E koahelang HLA-DRB101:01, HLA-DRB104:01, le HLA-DQ8, ho kenyelletsa tatelano e telele ea 13–17-mer e nang le litšobotsi tse netefalitsoeng tsa ho qhibiliha.
Ho ba le liphanele tse kantle ho sethala tse fumanehang bakeng sa dispatch acc hanghang
Bafani ba lokela ho fana ka li-CoA tse felletseng tse nang le: Peptide Therapeutic Supplier
le ho lokisoa ha nako ea ho boloka (mohlala, tekanyo ea iRT) bakeng sa LC-MS e matha.
Liphanele tsa QC tse Lebisitsoeng le Litifikeiti tse Feletseng tsa Tlhahlobo
Taolo ea Boleng bo matla (QC) Phanele bakeng sa li-peptide tsa referense tsa immunopeptidomics li tlameha ho fetela ka nģ'ane ho ponahalo ea boima ba MALDI-TOF.
Bafani ba lokela ho fana ka li-CoA tse felletseng tse nang le:
- Phaello e Phahameng ea ESI-HRMS Spectra: Ho netefatsa boima ba monoisotopic le linaha tsa tefiso ([M+H]^+, [M+2H]^{2+}, [M+3H]^{3+}) ka phoso e kgolo < 2 ppm.
- RP-HPLC Chromatograms: Ho bontša >95% kapa >98% bohloeki ba lik'hemik'hale bo nang le maemo a hlakileng a gradient.
- Boikemisetso bo setseng ba TFA: Ion Chromatography (KEA BONA) kapa 19F-NMR quantification e bontšang TFA e setseng < 1.0%.
- Net Peptide Content ka AAA: Litekanyetso tse nepahetseng tsa nanomoles/vial ho netefatsa hore li-spike-ins tsa ka hare li ka hlahisoa hape.
Bakeng sa Keletso: Ha o kopa li-peptide tsa SIL tsa tloaelo bakeng sa li-immunopeptidomics tse lebisitsoeng, kamehla etsa bonnete ba hore morekisi o fana ka AAA-certified net peptide content le TFA-mahala phapanyetsano ea letsoai. Mohato ona o le mong o felisa mohloli oa mantlha oa leeme la li-inter-batch quantification litlhahlobong tsa Orbitrap LC-MS..
Rapid Heavy-Isotope Labeling and Micro-Aliquoting Services
Bafani ba thepa ba tlameha ho nolofatsa motsoako oa li-peptide tse fetotsoeng tse rarahaneng, ho kenyeletsa:
- Automated Heavy Isotope Incorporation: Ho kopanya ka bokhabane bo phahameng ba [^{13}C₆, ^{15}N₂]Lys le [^{13}C₆, ^{15}N₄]Arg ntle le dilution ea isotopic nakong ea cleavage.
- Theminale & Liphetoho ka hare: Biotinylation, fluorophore conjugation (mohlala, FITC, Cy5), N-terminal acetylation, le li-click-chemistry handles (azide/alkyne) bakeng sa ho latedisa antigen ka mefuta e mengata.
- Ts'ebetso ea Kamore e Hloekileng: Bakeng sa li-bioassays tse sebetsang tse tlase (mohlala, Liteko tsa ts'ebetso ea T-cell, ELISpot, kapa liteko tse tlamang tsa TCR), li-peptide li tlameha ho sebetsoa tlas'a maemo a hloekileng a phaposi e hloekileng ka tlhahlobo ea endotoxin (< 0.01 EU/µg).
Tšebelisano 'moho: Ho phahamisa Immunopeptidomics Workflows
Ho sebetsana le mathata a li-immunopeptidomics tse lebisitsoeng ho hloka tumellano e haufi pakeng tsa baetsi ba lisebelisoa tsa reagent., lisebelisoa tsa mantlha tsa spectrometry, le balekane ba khethehileng ba peptide synthesis.
E le moetapele ea inehetseng oa ho etsa peptide, the MOL E fetola sethala sa peptide synthesis ea tloaelo e fana ka lisebelisoa tsa motheo tse kopantsoeng ka ho khetheha ho tšehetsa immunopeptidomics e tsoetseng pele le mananeo a ho sibolla neoantigen.
MOL e Fetola Enjene ea Immunopeptidomics
Phaello e Phahameng ea Phapanyetsano ea Letsoai ea SPPS Sehlopha sa AAA Quantitation Suite 100 Kamore e hloekileng
→ Motsoako oa Complex & Li-Peptide tsa Hydrophobic → TFA Counterion Removal (< 1.0% TFA e setseng) → Litaba tse netefalitsoeng tsa Net Peptide & Hantle Molarity → Endotoxin-Free & Ho beleha Lyophilization
Ka ho sebelisa chelete liphetoho tse phahameng tsa peptide le lits'ebeletso tsa ho tlosa TFA, Bafuputsi ba ka koala lekhalo lipakeng tsa boitokiso bo thehiloeng ho kit le boitšepo bo phahameng bo lebisitsoeng ho LC-MS.. Ho feta moo, tlhahiso ka hare nyopa Sehlopha 100 tlhahiso ea likamore tse hloekileng lisebelisoa li netefatsa hore maemo a maiketsetso a peptide hang hang a loketse tlhahlobo ea boima ba 'mele le netefatso ea immunology e thehiloeng liseleng..
Mehlala e tloaelehileng ea prep kits e fetotse ts'ebetso ea sampole ea immunopeptidomics. Ka ho kopanya lisebelisoa tsena le li-peptide tsa maiketsetso tse etselitsoeng bohloeki bo tiileng ba isotopic, Ho tlosoa ha TFA, le palo e netefalitsoeng ea AAA, bafuputsi ba ka theha matla, liphaephe tse phahameng tse tsamaisang ho sibolloa ha neoantigen le immunotherapy pele ka kholiseho e felletseng.
