05_tandem-mass-spectrometry

same AA compositions with exact same mass

-> AAs w. different AA masses (besides Leucine and Isoleucine)

reduction of mass by 18 Da through removal of H2O / peptide bonds

fingerprint of particular proteins through TOF measurement

-> peptide mass = part of a protein

consecutive stages of mass spectrometry with a chemical reaction in gas phase in btw. two stages

Ionisation -> mass analyser -> collision reaction cell -> mass analyser -> detector

typical experiment: MS1 followed by MS2

• MS1: isolation of most abundant ion for fragmentation

• MS2: readout

• next cycle: most abundant + 1

-> dynamic exclusion in time to prevent same peptides being analused over and over again

fragmentation around peptide bonds depending on which fragmentation method is used

=> exact computation of m/z and masses of peptide fragment ions from chemical and isotopic compostion

-> nomenclature for peptide fragments depending on bond break point and where charge is retained

Collision induced association CID/ Higher-energy collision dissociation HCD

1. nucleophile attack and cyclization

2. proton transfer

3. oxazolone formation

-> kinetic energy -> internal/vibrational energy => bond break

Electron transfer dissociation (ETD)

Dissociation of reagent ions/low energy electron/precursor ions

-> react cations w. electron donor -> charg ereduction -> change to radicals leading to fragmentation

• neutral losses (water, ammonia) -> satellite peaks in spectra

• satellite ions: sid chain cleavages

• internal ions: double backbone cleavage

• immonium ions: formed by simultaneous/sequential a- and y-type cleavage

Stage 1 - MS1

-> intact multiply charged peptides (almost always a mixture)

Stage 2 - MS2

-> fragments of singly and multiply charged peptides

=> different cutting positions resulting in different peak heights

“sequencing” by mass differences corresponding to AA masses

• starts w. C-term

• y1 may not be observed

• b1 almost never observed

• Leu/Ile isobaric

• different charge states appear in different m/z ranges

  -> deconvolution of charge state before sequencing

• by sequence tag database search and comparison through peptide mass, composition and sequence information

• spectrum matching

protein identification from partial peptide sequence

-> set of mass values combined w. AA sequence data to identify a peptide froma sequence M

required information

• peptide Mass M(r)

• m1: start mass

• partial sequence/tag

• m2: stop mass

• protein sequence database

• protease specificity

• modifications

• peptide and fragment tolerance

pros and cons

• rapid search times

• requires manual interpretation

• correct call required

peptide identification without manual interpretation from uninterpreteted tandem mass spectra

-> peptide-spectrum-match (PSM): matching of experimental and theoretical fragment ion lists

=> data base of known protein sequences required & only feasible for sequenced genomes

pros: easily automated for high throughput applications & matches for difficult interpretable spectra

cons: matches from marginal data

slow if:

• no use of enzyme specificity

• allowance of many variable modifications

• large database/dataet search