10.21.26 - The Cellular Proteome: Complexity Analyzed by Mass Spectrometry – Matthias P. Mayer

proteome, metabolome, and lipidome can all be measured by

mass spectrometry

basic principle of mass spectrometry

" generate ions, separate by mass-to-charge ratio (m/z), detect qualitatively and quantitatively by m/z and abundance"

"Parts of Mass Spectrometer and their functions"

ion source - ionization
mass analyzer - ion separation
detector - ion detection

what are the two main forms of ionization in MS

MALDI (Matrix Assisted Laser Desorption Ionization) and ESI (Electro-Spray Ionization)

What is the major difference between MALDI and ESI?

MALDI converts solid particles into gas, ESI converts liquid particles into gas

how do MS spectra resulting from MALDI and ESI differ and why?

ESI has many MS peaks over a large m/z scale (multiple charges) due to many proton transfers caused by explosions and collisions in final step

MALDI process

"

1. Analyte (peptides) imbedded into organic acid matrix 
2. Illumination by laser impulse which is absorbed by matrix 
3. Matrix heating and expansion (explosion into gas phase), carrying analytes 

"

ESI process

"

1. Steel capillary as anode (+), MS entry as cathode (-) 
2. Liquid with ions drawn into tailored cone 
3. Droplets containing solvent and positive ions discharged from steel capillary 
4. Solvent molecules evaporate, shrinking droplets and causing increased electrostatic repulsion in comparison to surface tension, explosion into droplets into gas 

"

What causes the different heights of peaks when reading peptide mass fingerprint?

"some peptides are more efficiently ionized than others"

What do the charges on an MS peptide peak represent?

"isotopes, seen as smaller equidistant peaks with a distance of (1/z)"

"Monoisotopic mass"

"sum of all exact isotopic masses of the most abundant isotope of the elements that make up a molecule "

"Average Mass formula"

"(sum of peak intensities x exact masses of peaks) / (sum of peak intensities) "

"relation of monoisotopic mass and average mass values"

"average mass always higher than monoisotopic mass "

mass resolution formula

"(mass of isotope peak) / (delta mass), with delta mass being the width of the peak at half intensity "

mass accuracy formula

"(experimental mass – true mass) / (true mass), in ppm"

Why is tandem mass spectrometry used?

"peptide mass fingerprint is not sufficient for unambiguous identification "

tandem MS principle

"Select individual molecule ions (precursor ion), induce fragmentation, analyze fragment spectra (product) "

parts of tandem mass spectrometer and functions

"

Ion source - ionization

Mass analyzer - ion separation 

Collision Cell – Fragmentation 

Mass analyzer - ion separation 

Detector - ion detection 

"

What are the three defined modes of fragmentation in TMS and their associated letters?

between alpha carbon and carboxyl - a,x
within peptide bond - b,y
between amino group and alpha carbon - c,z

What mode of peptide fragmentation is most common in collision induced/associated dissociation?

within peptide bond - b/y

"Product Ion Scan (TMS)"

"Select single m/z ion from sample, induce collision in cell, m/z scan products "

"Precursor Ion Scan (TMS)"

"Select one peptide after the other, induce collision induced dissociation, mass analyzer filters and lets past indicator ion with defined m/z "

"Neutral Loss Scan (TMS)"

"Both analyzers set to scanning mode with offset (as opposed to filter), see which peptides give rise to lost amount "

Which TMS scan(s) is(are) used to detect PTMs?

"Precursor Ion Scan, Neutral Loss Scan "

What are the two types of particle dissociation?

collision induced/associated and electron transfer

electron transfer dissociation

"chemical is charged with electron, collision causes transfer along backbone until cleavage occurs "

Why would you use electron transfer dissociation as opposed to collision induced dissociation?

not all peptides are efficiently cleaved by CID, such as long ones and those with PTMs

what are the steps of protein identification?

"Extraction, separation (gels), digestion (trypsin), peptide separation, sample ionization, MS, data analysis "

what information is matched up to identify proteins?

"information obtained from MS and information contained in protein and/or DNAseq databases "

probability based Mowse score formula

"-10Log(P)"

what must the Mowse score P value be greater than to be statistically significant? (non-random match)

70

what should the deviation of MS and calculated data masses be?

within instrument mass accuracy

what factors could cause a low probability score or peptide ambiguities?

"

Sample is not yet sequenced 

Imperfect sample separation 

Isoenzymes (closely related) 

PTMs (changing ion mass, reads as negative hit) 

Miscleavages 

Contamination with trypsin and keratin 

Mass accuracy low 

MS calibration 

Complexity of spectra increased due to salt adduct or fragmentation 

"

what are the two ways to use MS/MS to improve probability score?

autocorrelation and probability based matching

autocorrelation (MS/MS)

"comparison between theoretical MS/MS spectra of all peptides in database and experimental data "

probability based matching (MS/MS)

"comparison between theoretical MS/MS spectra of all peptides in database and experimental data, starting with most intense b and y peaks in top down method (y then b) "

what are some difficulties in detecting PTMs?

"

Transient 

Small fraction of peptides modified 

Multiple modifications within same protein/peptide 

In vivo degradation 

Labile during extract preparation 

Lost during sample workup 

Labile in mass spectrometer 

Not all peptides detected 

Increased search space 

"

what are some strategies for finding PTMs?

"

Compare modified and non-modified sample (measure MS sample peaks for before and after treating)

Search for indicator ions 

Data dependent neutral loss MS/MS/MS scan 

Enrichment of modified peptides 

"

two forms of labeling for quantitative proteomics

"SILAC (stable isotope labeling with amino acids in cell culture) and dimethyl labeling"

SILAC process

"

1. Two cell states (generally one normal and one supplied with heavy arg or lys isotopes) 
2. Combine lysates and purify proteins 
3. Digest and mass spec 
4. Peptides differ in mass, quantify levels from peak ratio 

"

dimethyl labeling process

"

1. Treat cells, lyse, and digest separately 
2. Separately label with different reagents (formaldehyde variants) 
3. Mass spec, Quantify levels from peak ratio 

"

what two tags are used for protein quantification using MS/MS?

"ITRAQ (isobaric tag for relative and absolute quantification) and TMT (tandem mass tag)"

how does MS/MS quantification work? (tags)

"both ITRAQ and TMT tags contain indicator ion and balance group
intial MS scan forms one peak of all samples
MS/MS scan shows indicator ion"

advantages of MS quantification

"

Label introduction early in sample preparation 

Quantification with several data points during elution 

"

disadvantages of MS quantification

"

Expensive 

Not possible with all samples 

Increased sample complexity limits multiplexing 

All peaks to be compared have to be sequenced by MS/MS 

"

disadvantages of MS/MS quantification

"Expensive

Introduction of label late during sample preparation 

Quantification only in single MS/MS spectra not during several elution points 

"

advantages of MS/MS quantification

"

Peptide ID and quantification within same spectrum 

High multiplex capacity 

No increase in sample complexity in MS 

"