32.17
Identificação de Peptídeos Usando Espectrometria de Massa Tandem
A espectrometria de massa em tandem, também conhecida como MS/MS ou MS2, é uma técnica analítica que emprega dois analisadores de massa. Essencialment…
Ao contrário do MALDI-TOF, a espectrometria de massa em tandem, ou MS/MS, emprega dois analisadores de massa colocados em série, separados por uma célula de colisão. Esta técnica pode identificar a sequência de aminoácidos de um peptídeo de uma mistura complexa para análise detalhada.
Primeiro, um ionizador por eletrospray gera íons peptídicos e os acelera para o primeiro analisador de massa como um quadrupolo. Este analisador atua como um filtro de massa, permitindo a seleção de um único íon peptídico chamado íon precursor de uma relação massa-carga específica.
Uma vez dentro da célula de colisão, o íon peptídico selecionado colide com as moléculas de gás, quebrando principalmente as ligações peptídicas e gerando íons menores.
Esses íons se movem para o segundo analisador de massa como um tempo de voo que separa os íons com base em sua relação massa-carga.
O espectro de íons do produto obtido ajuda a identificar a sequência de aminoácidos do peptídeo.
A combinação de MS/MS com cromatografia líquida auxilia na análise contínua de peptídeos de amostras biológicas complexas.
A MS/MS também pode caracterizar lipídios, identificar modificações proteicas pós-traducionais e detectar baixos níveis de biomarcadores específicos de doenças em amostras biológicas.
View the full transcript and gain access to JoVE Core videos
Q1: How does tandem mass spectrometry differ from single mass spectrometry?
Tandem mass spectrometry, or MS/MS, uses two mass analyzers in series separated by a collision cell, unlike single mass spectrometry. The first analyzer selects a specific peptide ion, which then fragments in the collision cell. The second analyzer separates the resulting product ions to build a fragmentation pattern, enabling amino acid sequence identification from complex mixtures.
Q2: What role does the collision cell play in tandem mass spectrometry?
The collision cell is positioned between the two mass analyzers and contains gas molecules that collide with the selected peptide ion. These collisions primarily break peptide bonds, generating smaller fragment ions. The resulting product ions then enter the second mass analyzer, where their mass-to-charge ratios are measured to determine the peptide's amino acid sequence.
Q3: How does liquid chromatography enhance tandem mass spectrometry analysis?
Combining MS/MS with liquid chromatography enables continuous peptide analysis from complex biological samples. Liquid chromatography separates peptides based on physical size and chemical properties before they enter the mass spectrometer, reducing sample complexity and improving the accuracy of peptide identification and sequencing.
Q4: What information does a product-ion spectrum provide about a peptide?
A product-ion spectrum displays the mass-to-charge ratios of fragment ions generated when a peptide breaks apart in the collision cell. This spectrum reveals the peptide's amino acid sequence by showing the pattern of fragments produced. Analysis software compares the obtained spectrum with protein databases to identify unknown peptides from overlapping fragments.
Q5: What are the main applications of tandem mass spectrometry beyond peptide identification?
Tandem mass spectrometry can characterize lipids, identify post-translational protein modifications, and detect low levels of disease-specific biomarkers in biological samples. In newborn screening, MS/MS analyzes dried blood-spot samples to detect inborn metabolic disorders like phenylketonuria and sickle cell disease, enabling early intervention and improved health outcomes.
Q6: How do hybrid MS/MS instruments improve analysis sensitivity?
Different analyzer combinations create hybrid MS/MS instruments that increase sensitivity and resolution. For example, quadrupole time-of-flight (QTOF) combines quadrupole and time-of-flight analyzers, while triple quadrupole instruments use two quadrupole mass analyzers separated by a collision cell. These configurations enhance the detection and characterization of peptides and other biomolecules.
Q7: How do bioinformatics tools help identify unknown peptides from MS/MS spectra?
Bioinformatics tools and protein database search algorithms compare the obtained MS/MS spectrum with databases of known peptide sequences. Since each protein produces a unique spectral pattern, the software matches overlapping fragments from unknown samples against known sequences, allowing researchers to identify proteins and peptides from complex biological mixtures.