Shotgun proteomikasida mikroplata sonikatsiyasi – Ilova eslatmasi
Shotgun proteomikasi murakkab biologik materialni LC-MS/MS-tayyor peptidlarga aylantirish uchun samarali, takrorlanadigan namuna tayyorlashga bog'liq. UIP400MTP mikroplastinka sonikatori kichik hajmdagi namunalarni standartlashtirilgan, parallel sonikatsiyani ta'minlab, laboratoriyalarga mikroplastinka asosidagi ish jarayonlarida buzilish, ekstraksiya va resolubilizatsiyani yaxshilashda yordam beradi. Ushbu ilova eslatmasida UIP400MTP proteomik namuna tayyorlashga qanday integratsiya qilinishi mumkinligi tushuntiriladi, bunda PLA2G12A/Th17 tadqiqotlaridan chop etilgan ekstrahujayralar-vezikula ish jarayoni laboratoriyada sinovdan o'tkazilgan misol sifatida ishlatilgan.
Ko'proq takrorlanadigan ov miltig'i proteomikasi uchun mikroplastinka sonikatsiyasi
Proteomika tadqiqotchilari uchun qayta ishlab chiqarilishi mumkin bo'lgan namunalarni tayyorlash yuqori sifatli LC-MS/MS maʼlumotlari uchun juda muhimdir. UIP400MTP mikrotasma sonikatori plastinka asosidagi va kichik hajmli/yukori o‘tkazuvchanlik ish oqimlarida standartlashtirilgan, parallell sonikatsiyani qo‘llab-quvvatlaydi.
U laboratoriyalar uchun ayniqsa foydalidir:
- Ko‘p quduqli namunalarda doimiy ishlov berishni talab qiladigan katta namuna to‘plamlari
- Namuna yo‘qotilishi va o‘zgaruvchanligini minimallashtirish lozim bo‘lgan cheklangan miqdordagi material
- Ekstrasellyulyar vezikulalar kabi lipidlarga boy namuna
- Samarali buzish, ekstraktsiya yoki qayta eritish zarur bo‘lgan murakkab biologik tayyorgarliklar
- Shartlar va replikalarda reproduktivlik muhim bo‘lgan solishtirma shotgun proteomikasi
Digestiyadan oldin mikrotasma sonikatsiyasini integratsiyalash orqali tadqiqotchilar namunaning tayyorligini yaxshilashlari mumkin:
- Oqsilni chiqarish va qayta eritish
- Tripsin/Lys-C hazm qilish
- Nano-LC/MS/MS olish
- Kvantitativ keyingi proteomika tahlili
Mikroplastinka sonikatsiyasi qo'lda o'zgaruvchanlikni kamaytirishga, namuna buzilishini yaxshilashga va murakkab biologik namunalarning ishonchli LC-MS/MS tahliliga tayyorlashga qanday yordam berishini o'rganing.
Mikroplastinka sonikatsiyasi quyidagilarga foyda keltirishi mumkin:
- Proteomika asosiy laboratoriyalari
- EV tadqiqot laboratoriyalari
- Immunologiya va hujayra-biologiya laboratoriyalari
- Translyativ tadqiqot guruhlari
- Hayot fanlari jamoalari, bitta namunadan yuqori o'tkazuvchan ish oqimlarigacha kengaytirishni rejalashtirayotganlar
The UIP400MTP is not an ultrasonic bath. It'a a high intensity cup horn for focused sonication. This powerful non-contact sonicator delivers a uniform sonication across all wells of your standard well-plate. You have precise control over amplitude, power, and pulsing. A built-in timer and temperature probe ensures consistent results. The UIP400MTP plate sonicator cools samples with water bath (external chiller optional).
The UIP400MTP is not an ultrasonic bath. It'a a high intensity cup horn for focused sonication. This powerful non-contact sonicator delivers a uniform sonication across all wells of your standard well-plate. You have precise control over amplitude, power, and pulsing. A built-in timer and temperature probe ensures consistent results. The UIP400MTP plate sonicator cools samples with water bath (external chiller optional).
Hujayra tashqi vesikul proteom tahlili uchun yuqori o'tkazuvchanlikdagi namunalarni tayyorlash
Shotgun Proteomika nima?
Shotgun proteomics has become a central analytical strategy for characterizing complex biological samples, from whole-cell lysates and tissue extracts to purified organelles, extracellular vesicles, and low-input clinical specimens. Its core strength lies in the coupling of protein digestion, high-resolution liquid chromatography-tandem mass spectrometry, and computational peptide-to-protein inference. However, the quality of the final proteome dataset is determined long before the sample reaches the mass spectrometer. Efficient sample disruption, protein solubilization, removal of interfering substances, reproducible enzymatic digestion, and robust peptide recovery are all decisive for depth of coverage and quantitative reliability.
Proteomika tadqiqotchilari va cheklangan yoki qimmatbaho namunalar bilan ishlayotgan hayotshunoslik laboratoriyalari uchun namuna tayyorlash ko'pincha to'siq bo'lib turadi.
UIP400MTP ishonchli namuna tayyorlash va mavjud laboratoriya ish oqimlari bilan oson integratsiyani ta'minlaydi.
Proteomikada ekstrasellulyar vezikulalar muammosi
Extracellular vesicles (EVs), for example, represent a particularly demanding sample class. They are membrane-bound, lipid-rich, nanoscale particles with relatively low protein yield and a high potential for carry-over of lipids, salts, detergents, serum proteins, and other matrix components. These features can interfere with digestion efficiency, chromatographic performance, electrospray stability, and peptide identification. A preparation workflow for EV proteomics must therefore be strong enough to disrupt vesicle structures and solubilize protein cargo, while remaining compatible with downstream enzymatic digestion and LC-MS/MS analysis.
Bu kontekstda, mikroplastinka bilan mos ultrasonikatsiya takrorlanuvchi, parallel namunalarni qayta ishlash uchun amaliy yondashuvni taklif etadi. Hielscher mikroplastinka sonikator modellari UIP400MTP (400 Vt) va UIP550MTP (550 Vt) ko'p quduqli plastinalarda va kichik hajmli idishlarda namunalarni sonikatsiya qilish uchun mo'ljallangan bo'lib, an'anaviy yagona zondli sonikatorlarga nisbatan yuqori o'tkazuvchanlikni qo'llab-quvvatlaydi. Proteomika laboratoriyalari uchun bu format jozibador, chunki u amaliy o'zgaruvchanlikni kamaytiradi, bir nechta namunalarni parallel ishlov berishni yaxshilaydi va plastinka asosidagi namuna tayyorlash quvurlariga tabiiy integratsiyalashga yordam beradi.
Namunali ish jarayoni
Yaqinda o'tkazilgan PLA2G12A asosidagi Th17-hujayra biologiyasida o'tkazilgan ekstrasellulyar vezikula proteomikasi ish jarayoni mikroplastinka sonikatori UIP400MTP miltiq proteomikasi namunalarini tayyorlashga qanday qo'shilishi mumkinligini laboratoriyada sinovdan o'tkazgan foydali misol sifatida taqdim etadi. Ushbu tadqiqot seriyasida EV namunalari metanol bilan davolash, UIP400MTP sonikatsiya, sentrifugatsiya, quritish, tripsin/Lys-C hazm qilish va nano-oqimli LC-yuqori aniqlikdagi tandem MS yordamida proteom tahlili uchun qayta ishlangan. Xuddi shu biologik ish dastlab bioRxiv preprint sifatida e'lon qilingan va keyinchalik Cell Reports jurnalida chop etilgan, unda proteomika tahlili PLA2G12A patogen T-hujayra javoblari kontekstida EV yuk oqsillarini qanday o'zgartirishini tavsiflashga yordam bergan.
Yükli oqsil ekstraktsiyasi uchun 96 teshikli plitalar sonikatori UIP400MTP
Sonikator: UIP400MTP mikroplastinkali sonikator
Kirish: 5 µg EV oqsili ekvivalenti
Haddelenish: Tripsin/Liz-C
O'qish: Nano-LC-MS/MS / DIA proteomikasi
Qadam-baqadam ko'rsatma: Shotgun proteomikasi uchun UIP400MTP yordamida EV namunasini tayyorlash
Ushbu ish oqimi UIP400MTP mikrotasa sonikatori yordamida kam miqdordagi ekstrasellulyar vakuol (EV) shotgun proteomikasi namunasi tayyorlash usulini tavsiflaydi. Bu nashr etilgan EV proteomika ish oqimiga asoslangan bo'lib, unda EV namunalar normalizatsiya qilinadi, quritiladi, metanol bilan ishlanadi, sonikatsiya qilinadi, tripsin/Liz-C bilan hazm qilinadi, kislotalanadi va nano-LC-MS/MS orqali tahlil qilinadi.
Ushbu protsedura proteomika tadqiqotchilari va hayotiy fan laboratoriyalari uchun mo'ljallangan bo'lib, EVlar yoki boshqa kichik hajmli, lipidlarga boy biologik namunalarni pastdan-yuqoriga shotgun proteomikasi uchun tayyorlashni nazarda tutadi.
Ish jarayoni umumiy ko'rinishi
| Bosqich | Maqsad | Asosiy natija |
|---|---|---|
| EVlarni tayyorlash | EV namunalari ajratiladi, yuviladi va miqdori aniqlanadi | Normalizatsiya qilingan EV miqdori |
| Namunaning quritilishi | Suvni eritmaning yordamida ishlov berishdan oldin olib tashlash | Quritilgan EV materiali |
| Metanol bilan ishlov berish | Lipidlarga boy EV materialini buzilishiga yordam beradi | Metanol bilan namlangan namuna |
| UIP400MTP sonikatsiyasi | Buzilish, ekstraktsiya va gomidlashuvni rag'batlantirish | Ishlangan EV namunasi |
| ovqat hazm qilish | EV proteinlaridan peptidlar hosil qilish | Tripsin/Liz-C peptidli digest |
| LC-MS/MS tahlili | Peptidlarni ajratish, aniqlash va miqdorini aniqlash | Proteomika ma'lumotlar to'plami |
| ma'lumotlarni tahlil qilish | Peptidlar va proteinlarni aniqlash va miqdorini aniqlash | Protein darajasidagi natijalar |
Qadam-baqadam protokol
| qadam | Ko'rsatma | Muhim parametrlari |
|---|---|---|
| 1 | Laboratoriyaning belgilangan izolyatsiya ish jarayonidan foydalanib tozalangan EV namunalarini tayyorlash. | Proteomika tayyorlashdan oldin hujayralar, chiqindilar va asosiy ifloslantiruvchilarni olib tashlash. |
| 2 | EV oqsil miqdorini BCA testi orqali miqdorini miqdoriy baholash. | Barcha namunalarni oqsilga teng kirishga normallashtirish. |
| 3 | 5 μg EV oqsil ekvivalentini toza PCR trubkalariga yoki mos past hajmli trubalarga o'tkazish. | Namuna yo'qolishi xavfli bo'lsa, past bog'lanishli trubkalardan foydalaning. |
| 4 | EV namunalarini to'liq quriting. | Qizib ketishdan saqlang; ko'rinadigan suyuqlik qolmaganini tasdiqlang. |
| 5 | Har quritilgan EV namunasiga 25 μL metanol qo'shing. | Quritilgan material to'liq namlanganiga ishonch hosil qiling. |
| 6 | UIP400MTP mikroplastinka sonikatori yordamida namunalarni 3 daqiqa sonikatsiya qiling. | Barcha namunalarda bir xil sonikatsiya sozlamalari va joylashuv mantiqidan foydalaning. |
| 7 | Sonikatsiyalangan namunalarni 19,000 × g da 20 daqiqa 4°C da sentrifuga qiling. | Sentrifugatsiyadan so'ng pellet yoki erimaydigan materialni bezovta qilmaslik. |
| 8 | Supernatantdan 20 μL ehtiyotkorlik bilan olib tashlang. | Barcha namunalar bo'ylab bir xil pipetlash usulidan foydalaning. |
| 9 | Qolgan namuna materialini to'liq quriting. | To'g'ridan-to'g'ri hazm qilishga o'ting yoki faqat tasdiqlangan sharoitda saqlang. |
| 10 | 4 μL tripsin/Lys-C eritmasini 100 ng/μL da 50 mM ammoniy bikarbonatda qo'shing. | Ferment eritmasini yangi tayyorlang yoki to'g'ri saqlangan aliquotlardan foydalaning. |
| 11 | Quritilgan oqsil materialini hazm eritmasida eritish uchun qisqa sonikatsiya qiling. | Sonikatsiyadan so'ng trubkaning tubidagi barcha suyuqlikni yig'ing. |
| 12 | 37°C da 2 soat davomida hazm qiling, 300 rpm da silkitish. | Qopqoqlarni qattiq yopiq tuting, bug'lanishning oldini olish uchun. |
| 13 | Hazm qilish uchun 1 μL 1.25% TFA qo'shiladi. | Kislotalanish hazm qilishni to'xtatadi va peptidlarni LC-MS/MS uchun tayyorlaydi. |
| 14 | Hazm qilishni LC-MS mos keluvchi flakon yoki plastinalarga o'tkazing. | Erimaydigan chiqindilarni o'tkazmaslik. |
| 15 | Nano-LC-MS/MS yordamida tahlil qiling. | In'ektsiya hajmi, LC gradienti va MS olish sozlamalaridan bir xil foydalaning. |
| 16 | Xom ma'lumotlarni DIA, DDA yoki maqsadli proteomika dasturiy ta'minotidan foydalanib qayta ishlang. | Mos ma'lumotlar bazasi, ferment spetsifikligi, o'zgartirish sozlamalari va FDR chegaralarini qo'llash. |
Ko'p quduqli plastinka sonikatori UIP400MTP
Nega mikroplastinka sonikatsiyasi?
The UIP400MTP enables standardized, parallel sonication of small-volume samples. This is useful when reproducibility, low sample input, and multi-sample throughput are important.
Use any standard microplate! High-throughput sample prep with the UIP400MTP
The referenced EV proteomics workflow used 5 µg protein-equivalent EV input, 25 µL methanol, 3 minutes of UIP400MTP sonication, trypsin/Lys-C digestion, TFA acidification, and nano-LC-MS/MS analysis.
Recommended LC-MS/MS and Data Analysis Steps
After digestion and acidification, samples can be analyzed using nano-flow reversed-phase LC coupled to high-resolution tandem mass spectrometry. In the published workflow, peptides were separated on a nano-LC system and analyzed by data-independent acquisition on an Orbitrap mass spectrometer.
| Bosqich | Recommendation | Maqsad |
|---|---|---|
| Sample loading | Use a C18 trap or equivalent peptide-loading setup. | Concentrates peptides and removes highly polar contaminants. |
| Peptide separation | Use nano-flow reversed-phase LC. | Improves peptide separation and MS sensitivity. |
| MS acquisition | Use DIA, DDA, or targeted MS depending on study design. | Generates peptide-level spectral data. |
| Database search | Use a species-appropriate reference database. | Supports peptide and protein identification. |
| FDR control | Apply peptide and protein false-discovery-rate thresholds. | Identifikatsiya ishonchliligini nazorat qiladi. |
| Miqdoriy aniqlash | Peptid, prekurzor va oqsil miqdori jadvallarini eksport qilish. | Guruhlar o'rtasida statistik taqqoslash imkonini beradi. |
Sifat nazorati tekshiruv ro'yxati
- Barcha namunalar bo'yicha EV oqsiliga teng kirishni tasdiqlash.
- Tasodifiy yoki muvozanatli namunaviy ishlov berish sxemalaridan foydalaning.
- Metanol hajmi, sonikatsiya vaqti, quritish vaqti va hazm hajmini bir xil saqlang.
- Peptid hosildorligi va umumiy oqsil identifikatsiyasini nazorat qiling.
- O'tkazib yuborilgan parchalanish tezligi va peptidni tekshiring uzunlik taqsimoti.
- Xromatografik cho'qqi shaklini va ushlab turish vaqtini takrorlashni tekshir.
- Ko'chmani nazorat qilish uchun bo'sh joylarni qo'shing.
- Katta tadqiqotlar uchun yig'ilgan QC namunalaridan foydalan.
- O'zgarishning takrorlanish koeffitsientini baholang.
- PCA yoki unga aloqador usullardan partiya ta'sirlari yoki chet namunalarni aniqlash uchun foydalaning.
Protokol tavsiyalari
Ushbu ish jarayonini nashr qilganda yoki hujjatlashtirganda, EV kirish miqdori, plitalar formati, metanol hajmi, UIP400MTP amplitudasi va sonikatsiya vaqti, markazlashtirish sharoitlari, quritish usuli, hazm qilish buferi, ferment konsentratsiyasi, hazm qilish vaqti, kislotalanish sharoitlari, LC-MS/MS platformasi, olish rejimi, dastur versiyasi, ma'lumotlar bazasi, FDR chegarasi, normallashtirish usuli va statistika ish jarayonini xabar qiling.
Barcha Hielscher mikroplata sonikatorlari integratsiyalashgan SD-karta orqali CVS fayli sifatida amplituda, sonikatsiya davomiyligi va harorat kabi muhim jarayon parametrlarini sana va vaqt bilan avtomatik yozib oladi. Takrorlanuvchanlik va sifat nazorati uchun ma’lumotlarni protokollash hech qachon bunday oson bo‘lmagan!
DIA proteomikasi uchun barcha namunalar bo'yicha izchil yig'ish sozlamalaridan foydalaning va imkon qadar birlashtirilgan QC namunalarini qo'shing. Prekursor soni, saqlash vaqti barqarorligi va replikatsiya o'zgaruvchanligini nazorat qiling.
Bu ish jarayoni EV proteomikasi uchun laboratoriyada sinovdan o'tgan misoldir. Boshqa namuna turlari uchun kirish miqdori, erituvchi sharoitlari, sonikatsiya vaqti, hazm hajmi va LC-MS/MS in'ektsiya strategiyasini optimallashtiring, so'ngra to'liq tadqiqotga o'tkaziladi.
Batafsil tadqiqot: PLA2G12A tadqiqotda EV Shotgun Proteomikasi
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In the EV preparation workflow, Th17 cells were cultured in medium containing exosome-depleted fetal bovine serum. Culture supernatants were first centrifuged to remove cells, filtered through a 0.22 µm filter, concentrated by ultrafiltration/ultracentrifugation, washed, resuspended in PBS, and quantified by BCA protein assay. This upstream EV preparation ensured that a defined protein-equivalent amount of EV material could be carried into the proteomics workflow.
For shotgun proteomic analysis, the authors used EV samples corresponding to 5 µg protein equivalents. These samples were dried in PCR tubes, and 25 µL methanol was added. The samples were then sonicated for 3 minutes using the UIP400MTP microplate sonicator. Following sonication, the samples were centrifuged at 4°C for 20 minutes at 19,000 × g. After removal of 20 µL supernatant, the samples were centrifuged to dryness. Proteins were then dissolved by sonication in 4 µL trypsin/Lys-C solution at 100 ng/µL in 50 mM ammonium bicarbonate. Digestion was performed for 2 hours at 37°C with shaking at 300 rpm. The digest was acidified with 1 µL of 1.25% trifluoroacetic acid and submitted to nano-flow LC-high-resolution tandem mass spectrometry.
This workflow highlights several useful principles for low-input EV proteomics. First, the input was normalized by protein equivalent, which is important when comparing EVs from different genotypes or treatments. Second, methanol was used before sonication, supporting disruption and extraction while avoiding detergent systems that may complicate LC-MS/MS. Third, the sonication step was short and standardized, which is compatible with parallel sample processing. Fourth, trypsin/Lys-C digestion was performed in a very small volume, reducing dilution and supporting low-input peptide recovery. Finally, direct transition from digestion to acidification and LC-MS/MS minimized unnecessary handling steps.
Pastki LC-MS/MS usuli nano-oqimli teskari faza ajratishni Q-Exactive HF Orbitrap mass-spektrometri bilan birlashtirdi. Namunalar C18 oldindan ustunida ushlab turilgan, so'ngra 50 μm ichki diametrli analitik ustunda 200 nL/min va 40°C da ajratilgan. Gradient past organik erituvchidan 35% erituvchi B gacha 60 daqiqa davomida o'tdi, so'ngra yuqori organik yuvish va qayta muvozanatlash amalga oshirildi. MS ni olish musbat-ion rejimida ma'lumotlarga bog'liq bo'lmagan yig'ilish va yuqori energiyali to'qnashuvli dissotsiatsiya yordamida amalga oshirilgan. DIA xom fayllari DIA-NN 1.8 yordamida in silico bashorat qilingan spektral kutubxona yordamida tahlil qilindi.
Yuqori o'tkazuvchan oqsil ekstraktsiyasi 96-quduqli plastinka sonikatori UIP400MTP bilan
tez-tez so'raladigan savollar
Shotgun proteomikada mikroplita sonikatsiyasidan kimlar ko'proq foyda oladi?
Mikroplita sonikatsiyasi, ayniqsa, bir vaqtning o'zida bir nechta namunalarni qayta ishlaydigan proteomika laboratoriyalari, jumladan, asosiy muassasalar, proteom tadqiqot guruhlari, immunologiya laboratoriyalari, translatsion tadqiqot jamoalari va yuqori o'tkazuvchan LC-MS/MS ish oqimlariga o'tayotgan hayotiy fan laboratoriyalari uchun juda foydali. Bu, ayniqsa, namuna-namuna takrorlanuvchanligi muhim bo'lgan hollarda dolzarbdir.
Odatiy proba sonikator o'rniga UIP400MTP ni nima uchun ishlatish kerak?
A conventional probe sonicator typically processes samples one at a time and requires careful cleaning between samples to reduce carryover. The microplate sonicator models UIP400MTP and UIP550MTP support parallel sonication in a plate-based or small-volume format, helping reduce manual handling, improve consistency, and streamline multi-sample preparation. They allow for easy integration into automated workflows, too.
Where does sonication fit into the shotgun proteomics workflow?
Sonication is usually applied during the pre-digestion sample-preparation phase. It can support disruption, extraction, homogenization, and resolubilization before enzymatic digestion with trypsin, Lys-C, or a trypsin/Lys-C mixture.
Bundan tashqari, sonikatsiya hazm qilish jarayonida ham qo'llanilishi mumkin, bu fermentativ oqsillarni sezilarli darajada tezlashtiradi. Sonikatsiya yordamida yuqori samarali oqsil hazm qilish imkoniyatlarini kashf eting, tez proteomik ish jarayoni uchun!
Mikroplastinka sonikatsiyasi ekstrasellulyar vezikula proteomikasi uchun foydalimi?
Ha. Ekstrasellulyar vezikulalar lipidlarga boy, membranaga bog'langan zarralar bo'lib, ularni takrorlash qiyin bo'lishi mumkin. Keltirilgan PLA2G12A tadqiqotlarida EV namunalari metanol bilan ishlangan, UIP400MTP bilan sonikatsiya qilingan, quritilgan, tripsin/Lys-C bilan hazm qilingan va nano-LC/MS/MS yordamida tahlil qilingan.
Qaysi namuna turlari mikroplastinka sonikatsiyasidan foyda ko'rishi mumkin?
Mikroplitalarni sonikatsiya qilish hujayra lysatlari, organell fraktsiyalari, immunopresipitatalar, protein agregatlari, membrana boy namunalar, ekstratsellyulyar vezikullar va boshqa past hajmli biologik tayyorgarliklar uchun foydali bo‘lishi mumkin. Har bir namuna turi sonikatsiya intensivligi va vaqti, erituvchi sharoitlari, kiritiladigan miqdor va hazm qilish strategiyasiga moslashtirilishi kerak.
Sonikatsiya fermentatik hazm o‘rnini bosadimi?
Yo‘q. Sonikatsiya namunani buzish, ekstraksiya yoki qayta eritish orqali hazm qilishga tayyorlashga yordam beradi. Pastdan-yuqoriga shotgun proteomikasiga peptide hosil qilish uchun proteolitik hazm qilish hali ham talab qilinadi.
Proteomika ish oqimlarida ultratovush yordamida protein hazm qilinish haqida ko‘proq o‘qing!
UIP400MTP past miqdordagi proteomika bilan mos keladimi?
Ha, mikroplata formati kichik hajmli ish jarayonlari uchun juda mos bo'lib, bunda namunalarni tejash va bir xil ishlov berish muhimdir. Nashr etilgan EV ish jarayonida proteomika tayyorlash 5 µg proteinga teng EV kirishidan amalga oshirilgan.
Mikroplata sonikatsiyasi takrorlanishni yaxshilashi mumkinmi?
Hielscher sonikatorlari bir nechta namunalar bo‘yicha standart sonikatsiya sharoitlarini qo‘llash orqali takrorlanishni ta’minlaydi. Biroq, hujayra yoki EV izolyatsiyasi, kirish normalizatsiyasi, hazm qilish samaradorligi, LC-MS/MS barqarorligi, ma’lumotlarni qayta ishlash va statistik tahlil kabi boshqa omillar ham proteomika takrorlanishiga hissa qo‘shadi.
Mikroplata sonikatsiyasi oqsil identifikatsiyasi chuqurligini yaxshilaydimi?
Namuna buzilishi yoki qayta eritish cheklovchi omil bo'lganda identifikatsiya chuqurligining yaxshilanishiga hissa qo'shishi mumkin. Ta'sir namuna turi, oqsil kimyosi, tozalash strategiyasi, hazm qilish shartlari va LC-MS/MS usuli samaradorligiga bog'liq.
Ushbu ish jarayonini muntazam ravishda ishlatishdan oldin nima optimallashtirilishi kerak?
Asosiy parametrlar qatoriga namuna miqdori, tampon yoki erituvchi tarkibi, plastinka formati, ultratovush amplitudasi va davomiyligi, quritish sharoitlari, hazm qilish hajmi, ferment konsentratsiyasi, inkubatsiya vaqti va LC-MS/MS injektsiya miqdori kiradi. To'liq tajriba to'plamiga ish jarayonini qo'llashdan oldin pilot sinov tavsiya etiladi.
Ushbu ish jarayonidan DIA proteomikasida foydalanish mumkinmi?
Yes. The referenced EV workflow used data-independent acquisition followed by DIA-NN analysis. Microplate sonication is part of the upstream sample-preparation process and can be integrated with DIA, DDA, or targeted proteomics methods.
What quality-control metrics should be monitored?
Recommended QC metrics include peptide yield, number of identified peptides and protein groups, missed-cleavage rate, peptide length distribution, chromatographic peak shape, retention-time stability, replicate coefficient of variation, carry-over, and separation of biological groups by PCA or related methods.
What is the main advantage of integrating the microplate sonicator models UIP400MTP or UIP550MTP into proteomics sample preparation?
The main advantage is standardized, parallel processing of small-volume samples. This helps laboratories reduce manual variability and prepare complex biological samples more consistently for digestion, LC-MS/MS acquisition, and quantitative proteomics analysis.
Hielscher microplate sonicators can be seamlessly integrated into automated workflows.
Adabiyot / Adabiyotlar
- FactSheet UIP400MTP Multi-well Plate Sonicator – Non-Contact Sonicator – Hielscher Ultrasonics
- FactSheet UIP550MTP Multi-well Plate Sonicator – Non-Contact Sonicator – Hielscher Ultrasonics
- Mochizuki-Ono C., Taketomi Y., Irie A., Kano K. et al. (2026): PLA2G12A-driven extracellular vesicle-lipid signaling amplifies pathogenic T cell responses in inflammatory diseases. Cell Reports 45, 2026.
- Mochizuki, Chika; Taketomi, Yoshitaka; Irie, Atsushi; Kano, Kuniyuki; Nagasaki, Yuki; Miki, Yoshimi; Ono, Takashi; Nishito, Yasumasa; Nakajima, Takahiro; Tomabechi, Yuri; Hanada, Kazuharu; Shirouzu, Mikako; Watanabe, Takashi; Hata, Kousuke; Izumi, Yoshihiro; Bamba, Takeshi; Chun, Jerold; Kudo, Kai; Kotani, Ai; Murakami, Makoto (2024): Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation. BioRxiv 2024.
- Lischnig A., Bergqvist M., Ochiya T., Lässer C. (2023): Corrigendum for “Quantitative Proteomics Identifies Proteins Enriched in Large and Small Extracellular Vesicles”. Molecular & Cellular Proteomics, 22; 2023.
- Lauren E. Cruchley-Fuge, Martin R. Jones, Ossama Edbali, Gavin R. Lloyd, Ralf J. M. Weber, Andrew D. Southam, Mark R. Viant (2024): Automated extraction of adherent cell lines from 24-well and 96-well plates for multi-omics analysis using the Hielscher UIP400MTP sonicator and Beckman Coulter i7 liquid handling workstation. Metabomeeting 2024, University of Liverpool, 26-28th November 2024.
- Cosenza-Contreras M, Seredynska A, Vogele D, Pinter N, Brombacher E, Cueto RF, Dinh TJ, Bernhard P, Rogg M, Liu J, Willems P, Stael S, Huesgen PF, Kuehn EW, Kreutz C, Schell C, Schilling O. (2024): TermineR: Extracting information on endogenous proteolytic processing from shotgun proteomics data. Proteomics. 2024.
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