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Mass Spectrometry Mass spectrometry

What is a Mass Spectrometer? Što je to mass spectrometer?

A mass spectrometer is based on the simple fact that different chemicals have different masses, and this is what determines what chemicals are present in a sample. A mass spectrometer je na temelju jednostavnog činjenica da različite kemikalije imaju različite mase, i to je ono što određuje što su kemikalije prisutne u uzorku. There are many types of mass spectrometers that not only analyze the ions differently but produce different types of ions; however they all use electric and magnetic fields to change the path of ions in some way. Postoje mnoge vrste masenim spektrometrima analizirati ne samo da su ioni drugačije nego proizvoditi različite vrste iona, no oni svi koristiti električna i magnetska polja kako biste promijenili put iona na neki način.

During the late 1980s and early 1990s, two new methods of sample ionization caused mass spectrometry to undergo a revolution in biopolymer analysis, namely ESI 38 and MALDI.39 Just over a decade ago, for the first time, mass spectrometric techniques that could measure the molecular masses of femtomole quantities of proteins in excess of 100 kDa, often to better than 0.01% accuracy, became widely available to protein chemists. Tijekom kasne 1980s i početkom 1990-ih, dvije nove metode uzorka ionization mass spectrometry prouzročio je prolaziti kroz revolucije u biopolymer analiza, naime ESI 38 i MALDI.39 Samo preko deceniju prije, po prvi put, mass spectrometric tehnikama koje mogu mjeriti s molecular masses of femtomole količine proteina veći od 100 kDa, često na bolje od 0,01% točnosti, postao je široko dostupna na protein apoteci. These methods are both successful in forming ions from large, labile biomolecules without significant degradation of the analyte. Te metode su uspješni u formiranju iona iz velike, labile biomolecules bez značajnije degradacije od analyte. Not only are they both sensitive techniques but also speedy – both MS and MS/MS sequence spectra can be acquired within seconds. Ne samo da su oba osjetljive tehnike, ali i brze - kako MS i MS / MS spektri slijed može steći u roku od sekunde. ESI and MALDI, due to their many differences, are complementary and many biopolymer laboratories have access to both techniques. MALDI i ESI, zbog njihove mnoge razlike, su komplementarni i mnoge biopolymer laboratorija imaju pristup i tehnike.

Sample preparation: 2-Dimensional Electrophoresis and Mass Spectrometry Primjer pripreme: 2 dimenzije elektroforeza i masena spektrometrija

Sample separation, isolation and preparation for the mass spectrometric techniques generally, involves 2-DE (2-Dimenisional Electrophoresis), a technique that can separate as many as 5000 different proteins in a single experiment. Uzorak separacija, izolacija i priprava za masovno spectrometric tehnika općenito, uključuje 2-DE (2-Dimenisional elektroforeza), tehniku koja može zasebne koliko 5000 različitih proteina u jednom eksperimentu. In general, 2-DE is capable of separating proteins within an isoelectric point (pI) range of 3.5–10 and of molecular masses ranging from 6 to 300 kDa, as well as being able to distinguish between post-translationally modified proteins (eg phosphorylated) and their non-modified companions. Općenito, 2-DE sposoban je odvajanje proteina u roku od jedne isoelectric point (PI) raspona 3.5-10 i molekularna masa u rasponu od 6 do 300 kDa, kao i biti u mogućnosti razlikovati post-translationally modificirani proteini (npr. phosphorylated ) I njihove ne-modified drugovima. Once separated, the protein components are revealed by staining techniques (eg silver stain, fluorescent stain, Coomassie blue) and once located they can then be extracted from the gel. Nakon što se odvojili, protein components se objavi po staining tehnike (npr. srebrna boja, fluorescentna mrlja, Coomassie plava) i kada se nalaze oni tada mogu biti preuzete iz gela. Proteins cannot easily be eluted from gels without the use of detergents, which are detrimental to the mass spectrometric analysis; additionally, large proteins tend to be heterogeneous (eg as a result of glycosylation) and so possesses no single molecular mass which can be related to the corresponding entry in a database. Proteini ne može jednostavno biti eluted iz gelova bez deterdženata, koje su štetne za masovno spectrometric analysis; dodatno, veliki proteini imaju tendenciju da se heterogena (npr. kao rezultat glycosylation) i na taj način ne posjeduje jednu molekularna masa koja se može se odnose na odgovarajući unos u bazu podataka. To overcome these obstacles, the elution step tends to be accomplished after the protein has been digested by an aqueous acetonitrile wash of an excised gel piece. Da biste prevladali prepreke te, elution korak teži da se nakon protein je digested by jednom voden acetonitrile opere u jednom komadu excised gel. This increases the yield of extraction 5 and by using an in-gel digestion method employing trypsin, which has been described and is widely used as published or with minor modifications, produces a MS-compatible sample from which a protein identification can be made. Ovo povećava prinos od 5 ekstrakcija i putem in-gel probavu trypsin method zapošljavanja, što je i opisano je naširoko koristi kao objavljenih ili uz manje izmjene, proizvode MS-kompatibilan uzorku od kojih jedan protein identifikacija može biti. The digestion step can be preceded by an optional reduction and alkylation step to cleave and block disulfide bridges that exist between cysteine residues. The probavu korak može biti prethodi opcionalna redukcija i alkilacija korak do prionuti i blok disulfide mostova koji postoje između cysteine ostataka. Robotic systems have been developed to automate the excision of protein spots from the 2D-gel, to carry out the subsequent enzymatic digestion and to transfer the samples onto MALDI-MS target plates for the initial MS analysis. Robotika sistema su razvijeni kako bi automatizirati excision of protein spotova iz 2D-gel, izvršiti naknadne enzimska probavu i za prijenos uzorci na MALDI-MS ciljne ploče za početno MS analysis. For many applications, the peptides recovered from in-gel digestions require concentration and purification before being analysed by mass spectrometry especially if ESI is the ionization method used. Za mnoge aplikacije, peptida oporavila od in-gel digestions zahtijevaju koncentraciju i pročišćavanje prije nego se analiziraju by mass spectrometry posebno ako ESI je ionization method used. Reversed phase high performance liquid Reversed phase high performance liquid
chromatography (HPLC) is one method of achieving this; another method involves the use of ZipTips (Millipore) (pipette tips packed with C18 material) or Poros R2 perfusion material (Perseptive Biosystems). kromatografija (HPLC) je jedan način postizanja ovog; druge metode uključuje korištenje ZipTips (Millipore) (pipette savjete pakiran sa C18 materijal) ili Poros R2 perfuzijske materijal (Perseptive Biosystems). Despite its widespread acceptance, the limitations of 2-DE include the exclusion of very small or very large proteins, very acidic or very basic proteins, as well as hydrophobic proteins such as membrane proteins. Unatoč rasprostranjenog prihvaćanja, ograničenja od 2-DE uključuju izuzeća od vrlo male ili velike proteini, vrlo kiselinski ili vrlo osnovnih proteina, kao i hydrophobic proteina, kao što su membranski proteini. It is thought that To je mislio da
only 20% of the gel-loaded proteins can be visualised and analysed. samo 20% od gel-učita proteina može biti visualised i analiziraju. Other constraints are that the amount of sample that can be loaded is limited causing the non-observance of Ostali uvjeti su da je količina uzorka koja može biti učitan ograničen je uzrok ne-pridržavanje,
low concentration proteins and also that the most commonly used staining techniques have non-linear response factors. niska koncentracija proteina i da se najčešće koriste staining tehnike imaju nelinearni odziv faktora. In practice, 2-DE is a labour intensive process involving manual handling steps that offer the opportunity for impurities such as keratin to contaminate the samples. U praksi, 2-DE je radni proces koji uključuje intenzivne priručnik za rukovanje korake koje nude mogućnost za nečistoća kao što je keratin se zaraziti uzorci. One 2-D gel will take a day to complete and about one month for a complete structural analysis by MS, although automation can help both the contamination problem and speed up the analysis to some extent. Jedna 2-D gel će se jedan dan kako bi upotpunili i oko jedan mjesec za kompletnu strukturna analiza od strane MS, iako automatizacija može pomoći i onečišćenje problem i ubrzati analizu do određene mjere.

Alternative protein separation techniques Alternativna protein separation tehnika

Alternative, MS-compatible methods of protein preparation are under development but no one technology has emerged as a universal replacement. Alternativna, MS-kompatibilnim metodama protein pripremi su pod razvoj, ali nitko ne tehnologija je nastao kao univerzalnu zamjenu. These methods aim generally to interface the protein sample directly to MS/MS analyses thereby eliminating time-consuming sample preparation steps and the need for a preliminary MS analysis. Te metode imaju za cilj da sučelje općenito proteina uzorka izravno na MS / MS analize time eliminira dugo trajati uzorak pripreme korake i potrebu za idejno MS analysis. Capillary isoelectric focusing (CIEF)-MS and preparative isoelectric focusing followed by size exclusion chromatography-MS are methods that have been compared in depth with 2-DE in terms of separation effciency, peak capacity, precision and robustness, with the former appearing the more favourable alternative.The direct analysis of complex peptide mixtures from a mixture of proteins using on-line, reversed phase high performance liquid chromatography (HPLC)-MS/MS has been used successfully as an alternative to 2DE and this has led onto multidimensional chromatography if greater peptide separation is desirable prior to the MS/MS analyses. Kapilara isoelectric fokusiranje (CIEF)-MS i spremanje isoelectric fokusiranje slijedili po veličini iskljucivanja kromatografija-MS su metode koje su u dubini u usporedbi s 2-DE u smislu razdvajanja effciency, peak kapaciteta, točnost i robusnost, s bivše pojavljuju u više povoljni alternative.The direktni analiza kompleksnih mješavina peptida iz mješavina proteina pomoću on-line, preinačio phase high performance liquid chromatography (HPLC) -MS/MS je uspješno koristi kao alternativa 2DE i ovo je vodio na višedimenzionalno kromatografija ako veći peptida razdvajanja je poželjno prije MS / MS analize. Examples of two dimensional chromatography include anion or cation exchange followed by reversed phase HPLC and size exclusion chromatography followed by reversed phase HPLC. Primjeri uključuju dva dimenzionalan kromatografija anion ili cation exchange a nakon toga je preinačio phase HPLC i veličina iskljucivanja kromatografija nakon toga je preinačio phase HPLC. Another, alternative approach has been to use combined solid-phase micro-extraction together with capillary electrophoresis(CE) coupled to ESI MS/MS for the analysis of a total protein tryptic digest. Drugi, alternativni pristup je koristiti u kombinaciji solid-phase mikro-ekstrakcija, zajedno s kapilarna elektroforeza (CE) spojenih na ESI MS / MS za analizu ukupno protein tryptic digest. This method afforded high resolution separation of the peptide fragments allowing the identification of 80–90% of the proteins in this particular yeast ribosome complex. Ova metoda afforded visoke rezolucije odvajanje od peptida fragmenti omogućava identifikaciju 80-90% proteina, u ovom konkretnom kvasac Ribosomi kompleksa. Coupling microfluidic devices to MS is a further strategy that combines sample handling and separation, as well as interfacing neatly with nano-ESI-MS analysis.A different approach for selective protein fractionation and identification uses ProteinChip technology (Ciphergen) which employs a surface capture method using antibodies or chemically modified surfaces to capture specific classes of proteins which are then analysed by MALDI-MS. Coupling microfluidic uređaja na MS je daljnja strategija koja kombinira uzorak rukovanje i odvajanje, kao i jednostavna sučelja s nano-ESI-MS analysis.A drugačiji pristup za selektivno protein fractionation i identifikacije koristi ProteinChip tehnologija (Ciphergen) koja zapošljava površine zauzimanja metoda pomoću protutijela ili kemijski modificirane podloge za hvatanje određene klase proteina koji su zatim analiziraju by MALDI-MS. This technique, known as Surface Enhanced Laser Desorption Ionization (SELDI)-MS35, has great potential for the comparison of the protein content of different samples. Ova tehnika, poznata kao Površina poboljšane laserska desorpcija ionization (SELDI)-MS35, ima veliki potencijal za usporedbu, protein content različitih uzoraka.