Samenvatting
Summary lectures Protein dynamics and metabolomics
Detailed summary of the lectures of the course 'Protein dynamics and metabolomics'
[Meer zien]Voorbeeld 4 van de 35 pagina's
Voorbeeld 4 van de 35 pagina's
In winkelwagengesponsord bericht van onze partner
Voorbeeld van de inhoud
Lecture 1 - Introduction to mass spectrometry basedproteomics (02-09)
maandag 2 september 2019 15:14
MS based proteomics: can identify the proteins inside the cells
All the 21.000 genes are not expressed at the same time. The body has around 150 cell types,
each cell type expresses about 10.000 proteins.
Genomics (next-generation sequencing) --> Can read DNA and RNA sequences (nucleotide
sequences)
- Entire genomic sequence
- Expressed genes ((RNA)
- DNA interactions
Proteomics (mass spectrometry) --> Can read amino acid sequences (proteins)
- Expressed proteins
- Protein complexes
- Post-translational modifications
Systems biology combines genomics, proteomic and computational biology.
Main proteomics research aims:
1. Use the technology to identify the large proportions of the proteome
2. Quantify the proteins
3. Identify protien modifications
4. Characterize protein interactions
2D gels
- Power compared to bottum-up proteomics: Different forms of the same proteins (e.g.
splice variants, PTMs) appear in different spots
- Limitations
○ Poor reproducibility
○ Poor dynamic range
○ Poor coverage (max. 1000 protein identifications)
○ Difficult quantification of proteins
○ Problems to resolve highly acidic/basic proteins, or proteins with extreme size of
hydrophobicity
○ Difficulties in automation --> very low throughput
--> 2D gels are hardly used anymore
General workflow for mass spectrometry-based proteomics
Cells --> Lysis --> Digestion--> HPLC ---> MS --> Full scan --> read proteins
PTMs = post translational modifications
PPI= protein-protein interactions
The complexity of the human proteome
- 10.000-12.000 proteins expressed per cell/tissue type
○ Tryptic digestion results in about 500.000 peptides
--> The transcriptome (all mRNA expressed inside cells) spans 4 orders of magnitude in
abundance. A proteome spans 6 (cell line) to 9 (plasma) orders of magnitude.
Difference in using high and low abundant orders causes high challenges.
GENOMICS - Next Generations Sequencing
Lectures Pagina 1
,GENOMICS - Next Generations Sequencing
Pro's:
- Comprehensive detection of the transcriptome
- Quantitative
- Single-cell techniques available
- Technique very well developed
Con's:
- Transcriptome does not correlate very well with the proteome; The quantitiy of mRNA
does not always say something about the quantity of proteins
PROTEOMICS - Mass spectrometry
Pro's:
- Proteome represents the 'celluar machines' (also PTM and PPI)
--> Answer to what is going on in the cell
Con's:
- Not inherently quantitative
- Not comprehensive yet (not detected does not mean 'not present')
- Not possible for single cells (yet)
- Still a young technique
General workflow for bottum-up/shotgun MS-based proteomics
1. Grow cells or organisms containing proteins
a. Grow in culture dish or isolate from organism
b. Everything that contains proteins can be used
2. Proteins will be lysed to be extracted
3. All these proteins will be digested by a protease to end up with smaller peptides to use for
MS
a. The protease predominantly used is Trypsin; robust and active enzymes which
cleaves at the C-terminal
4. All these peptides are loaded on a HPLC to load the MS gradually
RP-HPLC: reversed phase high performance liquid chromatography
Over time fractions of the peptide will elute gradually, in which they can gradually enter the MS.
- Peptides bind to C18 matrix in hydrophilic acidic solvent (e.g. 0.5% acetic acid)
- Peptides elute with increasing concentration of organic solvents (e.g. acetonitrile)
--> Peptides are separated according to their hydrophobicity
--> The thickness of the LC column is of importance for the elution time and the volume of the
elution. If you use a smaller column, you get a shorter and intenser signal.
--> Causes gradually entrance of the digested peptide fractions into the Mass spectrometer,
because each fraction elutes over another time window.
Nano reversed phase ultra high performance liquid chromatography (nano RP-UHPLC)
At the tip of the needle, tryptic peptides wil come out as liquid droplets, solvent solution
evaporates. After this the process electrospray occurs.
Electrospray ionization: during gradually evaportaion of the droplets, the charge gets higher. At
a moment the tension at the surface gets so high, that it will explode. Eventually you end up
with positively charged ions that are in the gass phase and get sucked into the MS.
3 basic components in a mass spectrometer:
1. Ionization source
2. Mass analayzer
--> Mass analyzers meaur the mass-to-charge (m/z) ratios of gas phase ions in order to
separate them from each other
--> Each analyzer has its own strengths and weaknesses; most mass spectrometers use two
or more mass analyzers
3. Detector
Mass resolution: how accurately you are measuring a certain mass that you are interested in
Lectures Pagina 2
,Mass resolution: how accurately you are measuring a certain mass that you are interested in
Importance to measure with very high resolution: having the ability to separate two molecules
with a slight difference mass, you will get two difference peaks. If you measure those with a low
resolution, you will only get one peak.
--> Scanning at higher resolution costs a lot of time, therefore it is of importance that you find
and equilibrium between a good resolution where you can measure a lot of peptides, as fast as
possible.
Overview of mass analyzers
- TOF: Time of flight
○ High speed
○ High sensitivity
○ High dynamic range
○ Medium resolution/mass accuracy
--> Small molecules move fast, larger molecules move slowly
- Quadrupoles and iontraps: Contains metal rods, starts by injecting peptides into iontraps.
By changing voltages you can either trap the peptides or let them go through to the
analyzer.
○ High speed
○ High sensitivity
○ Cheap
○ Low resolution
○ Low mass accuracy
- Orbitrap
○ High resolution
○ High mass accuracy (sub ppm)
○ High dynamic range
○ High sensitivity
○ Low speed
Orbitrap mass analzyer
- Ions are electrostatically trapped in an orbit around a central, spindle shaped electrode
(--> no magnet needed)
- A image current is induced by pasing by ions at the detector plates
- M/Z is determined by Fourier Transformation (transformation of the frequency to a
peptide signal with a certain mass) of the periodic signal
- High sensitivity and high resolution
- Most dominant mass analyzer in modern proteomics
--> Different molecules starts occylating creating a frequency for each molecules, which will be
translated in a peptide signal with a certain mass.
--> Masses are recorded with an accuracy below 1 parts per million (ppm)
In an orbitrap: frequency of molecules are translated into masses.
Masses from an orbitrap are measured with a very high accuracy.
Due to the big data you obtain, it is impossible to handle it manually. This is done by the
software QExactive.
High resolution MS visualizes a so-called isotope cluster: this is due to the different isotopes of
each Carbon/hydrogen/oxygen/nitrogen molecule.
Peptide charge following Electrospray ionization (ESI)
Tryptic peptides are primarily doubly charged (but other charge states are present)
Higher charges (3+ and higher are possible, e.g.:
- Missed cleavage (internal R/K)
- PTMs
- Histidine side-chain
Lectures Pagina 3
, Measured value in mass spectrometer is mass (m) divided by charge (z) of the peptide (=number
of attatched protons) = m/z
--> From the isotope cluster you can decude the charged state, we need that calculation to get
to the mass of the corresponding peptide
--> Without the isotopic cluster you cannot calculate the mass of the molecule
Calculating the mass of a peptide
By known the charged state you can easily calcute the mass of a peptide.
The charged state can be deduced from the mass/charge. You multiply the monoisotopic peak
by the charged state, then subtract the amount of protons. Than you have the mass in Da.
Fragmentation of peptides
Fragmentation occurs mainly on the peptide backbone; results mainly in b (N-terminal
fragment) and y ions.
--> Peptide sequence can be read out solely by using the accurate precursor peptide mass and
the fully annotated MS/MS spectrum (de novo peptide sequencing)
--> Combination of very accurate peptide precursor mass and part of the fragment spectra still
allows idenfication by doing a database search against theoretical peptides/spectra
The fragmentation spectra allows you to identify (parts of) the amino sequence by the peptides
you fragmented.
With knowlegde from the database search, you sometimes do not need full coverage of a
protein to identify it.
Thousands of proteins (and their PTMs) can be identified with just a few hours of measurement
time and a few micrograms of protein, and a very good and automated software!
Lectures Pagina 4
Voordelen van het kopen van samenvattingen bij Stuvia op een rij:
Verzekerd van kwaliteit door reviews
Stuvia-klanten hebben meer dan 700.000 samenvattingen beoordeeld. Zo weet je zeker dat je de beste documenten koopt!
Snel en makkelijk kopen
Je betaalt supersnel en eenmalig met iDeal, creditcard of Stuvia-tegoed voor de samenvatting. Zonder lidmaatschap.
Focus op de essentie
Samenvattingen worden geschreven voor en door anderen. Daarom zijn de samenvattingen altijd betrouwbaar en actueel. Zo kom je snel tot de kern!
Veelgestelde vragen
Wat krijg ik als ik dit document koop?
Je krijgt een PDF, die direct beschikbaar is na je aankoop. Het gekochte document is altijd, overal en oneindig toegankelijk via je profiel.
Tevredenheidsgarantie: hoe werkt dat?
Onze tevredenheidsgarantie zorgt ervoor dat je altijd een studiedocument vindt dat goed bij je past. Je vult een formulier in en onze klantenservice regelt de rest.
Van wie koop ik deze samenvatting?
Stuvia is een marktplaats, je koop dit document dus niet van ons, maar van verkoper brittleeden. Stuvia faciliteert de betaling aan de verkoper.
Zit ik meteen vast aan een abonnement?
Nee, je koopt alleen deze samenvatting voor €4,99. Je zit daarna nergens aan vast.
Is Stuvia te vertrouwen?
4,6 sterren op Google & Trustpilot (+1000 reviews)
Afgelopen 30 dagen zijn er 53068 samenvattingen verkocht
Opgericht in 2010, al 14 jaar dé plek om samenvattingen te kopen