Proteomics Introductions#
Lennart Martens - Introduction to Proteomics#
Digitalizer: Transform an analog signal into a digital signal
Ionization Types
MALDI: Analyte (peptide) is charged by matrix molecules by one ion only (mechanism is not so clear)
three common matrix molecules in proteomics (CHCA, SA, DHB): Benzeme group taking up laser energy. Inherent bias to three amino-acids by MALDI due to three amino-acids sharing conformational similarity with benzeme group
Electrospray ionization (ESI): Acid is added to the liquid in order to obtain charged peptides.
temperature of the needle
peptides can take more than one ion
roughly 150.000 possible peptides if 5000 genes are expressed in a tissue (without any PTMs)
Detectors get worse over time. In the maintence sample one should see a spike when a new detector is replaced for the old one.
Fragmentation of peptides
collision-induced dissociation (CID): b and y ions
ergodic process, continously stronger vibration of peptide cleaves PSMs
electron-capture dissociation (ECD): c and z ions
preserves PTMs as the fracturing is non- ergodic (vibration induced)
Intro (OpenMS from Tübingen)#
Ion mobility as a way to identify number of charges (ions), adding another dimension to the data
predict MS2 peptide itensities in order to better identify peptides (MS2PIP, DeepMass: Prism, Prosit)-
Question: Does this take amino-acid sequences and provides spectra?
number of mappings from peptides to protein (How many peptides per peptide?)
absoute quantification siscpa, aqua
feature-based label-free quantification
does scale to (100?)
quantification of isotopes (3D integral: intensity over retention time and m/z )
SWATH-MS: DIA in DDA setting?
HUGO PSI Standards Formats: Machines do not provide all the same standardized dataset.
KNIME is popular for custom machines.
Nextflow
for cloud providers
Mass Spectrometry#
Unbiased analysis that does not require prior knowledge of the sample composition
Analytical technique which identifies molecules based on their mass and charge (m/z)
Proteomics: the large-scale study of proteins.
Pipeline#
Liquid Chromatorgraphy (LC)#
Peptide separation by hydrophobicity
hydrophilic vs hydrophobic liquids (Acetonitrile)
Column#
reverse phase (chromatography) (also called RPC, reverse-phase chromatography, or hydrophobic chromatography) includes any chromatographic method that uses a hydrophobic stationary phase. RPC refers to liquid (rather than gas) chromatography.)
Reversed-phase chromatography (also called RPC, reverse-phase chromatography, or hydrophobic chromatography) includes any chromatographic method that uses a hydrophobic stationary phase. RPC refers to liquid (rather than gas) chromatography. (…) Reversed-phase chromatography is a technique using alkyl chains covalently bonded to the stationary phase particles in order to create a hydrophobic stationary phase, which has a stronger affinity for hydrophobic or less polar compounds. The use of a hydrophobic stationary phase is essentially the reverse of normal phase chromatography, since the polarity of the mobile and stationary phases have been inverted – hence the term reversed-phase chromatography.
75um ID packed with 3um/1.9um reverse phase C18 beads. Pulled fused silica
Column performance is very important for your experiments.
If the column is not packed perfectly you will have dead volumes and peak tailing.
You will pick the same peptides for identification
Mass Specometry#
“One of the most significant differences between transcriptomics and proteomics is in the dynamic range of mRNA and protein concentrations inside the cell. While the protein abundances stretch over at least seven orders of magnitude, from one copy per cell to ten million copies per cell, the mRNA dynamic range covers only three or four orders of magnitude.” (https://doi.org/10.1002/pmic.201200451)
Claim: Around 5000 proteins should be identified for each sample.
Data Dependent Acquistion (DDA)#
Orbitrap specific steps:
MS1: mix of peptides to identify most candidates for MS2 scan
MS2: one peptide (z/m ratio) which is then fragmented and scanned
Default: 12 MS2 and 1MS1 scan in parallel
Peptide Identification#
How do we get from acquired spectra to protein and peptide identifications?
some peptides have the same mass
To identify peptides the mass spectrometer performs a fragment (MS2) scan on an isolated peptide ion
peptides with the same m/z ratio are fragmented and then analyzed (“de novo” sequencing)
Amino Acids and residuals#
Name |
abr |
code |
Residue Mass |
---|---|---|---|
Alanine |
Ala |
A |
71.03711 |
Arginine |
Arg |
R |
156.10111 |
Aspartic Acid |
Asn |
N |
114.04293 |
Cysteine |
Cys |
C |
103.00919 |
Glutamic Acid |
Glu |
E |
129.04259 |
Glutamine |
Gln |
Q |
128.05858 |
Glycine |
Gly |
G |
57.02146 |
Histidine |
His |
H |
137.05891 |
Isoleucine |
Ile |
I |
113.08406 |
Leucine |
Leu |
L |
113.08406 |
Lysine |
Lys |
K |
128.09496 |
Methionine |
Met |
M |
131.04049 |
Phenyalanine |
Phe |
F |
147.06841 |
Proline |
Pro |
P |
97.05276 |
Serine |
Ser |
S |
87.03203 |
Threonine |
Thr |
T |
101.04768 |
Trypthophan |
Trp |
W |
186.07931 |
Tyrosine |
Tyr |
Y |
163.06333 |
Valine |
Val |
V |
99.06841 |
Residue mass is referring to the mass in an peptide of a amino acid
Confunding Factors (or Hyperparameters)#
Critical parameters for DDA methods. Recommendation for machines at CPR
Max Injection Time
The maximum time which the instrument will use to reach the target amount of ions in the C-trap
Low max injection times gives faster scans speed.
High max injection times gives better intensity and dynamic range
Automatic Gain Control (AGC) Target
The target ion amount which will be accumulated in the C- trap
A higher AGC target will give higher intensity
Dynamic Exclusion Time
The time which the instrument will exclude precursors already selected for MS2.
Exclusion time is dependent on the length of your gradient.
For 145 minutes, we usually have 30 seconds
Number of MS2 scans (Top N)
More MS2 scans gives deeper protein coverage but slower speed.
sample overloading (too much liquid) messes up scan (relation to Dynamic Range?)
dwell time
the time a particular ion (m/z) signal is monitored
cycle time
Techniques#
TOF
ORBITRAP
PASEF
FAIMS
HCD Cell
Orbitrap#
Amino Acid weights#
fragments of peptides are identified on their weights
Glossar#
Term |
meaning |
---|---|
c-Trap |
Meaning of c? collects one million particles before forwarding them |
elute |
remove (an adsorbed substance) by washing with a solvent, especially in chromatography |
HCD |
|
XIC |
Extracted Ion Current |