1. Tandem MS (MS/MS) on the Q-ToF2

2. On-line LC-MSMS on the Q-ToF2 : peptides from a single protein
Separate peptides over a 60 minute LC run, perform tandem MS on each peptide
Fragment Mass spectrum (MSMS):
fragments from one peptide
Chromatogram
Survey Mass Spectrum - intact peptides
mass/charge
Information from an MSMS experiment with peptide separation on the CapLC
with the separated peptides being fed directly in-line to the Q-ToF
Mass infomation about the intact peptide and the fragments – lot more info to search with
time

3. Tandem MS - peptide de novo sequencing
low energy collision fragments the peptide
cleavage usually occurs at the amide bond i.e. between residues
series of peptide fragments
each fragment is one amino acid longer than the next
the series of fragments corresponds to the sequence of the peptide
During low energy collisions in the Q-Tof, the most facile cleavage of the peptide occurs at the amide bond which leads predominantly to the formation of y type ions (retention of the charge at the C-terminal side) and some low molecular weight b type ions (retention of the charge at the N-terminal side).
The range of different fragment ion masses present in the MS/MS spectrum may then be attributed to part or occasionally all of the peptide sequence.
each peptide fragment in a series differs from its neighbour by one amino acid.
possible to determine the amino-acid sequence by considering the mass difference between neighbouring peaks in a series
However, the difficulty lies in the fact that the information in tandem-MS spectra is often not complete and that intervening peaks, which might or might not belong to the series, can confuse the analysis. For example, a mass difference of 114 Da might be found between two large peaks, but a very small peak might also be found at 57 Da between these two large peaks. This part of the spectrum could therefore correspond to one asparagine (residue mass = 114 Da) or two glycines (residue mass = 57 Da). In practice, experts can correctly interpret at least parts of tandem-MS spectra, whereas computer algorithms are, as yet, unreliable for determining amino-acid sequences. In

4. peptide de novo sequencing
How does amino acid sequencing on a mass spec work? Again the msms procedure is used
The process depends on the low energy collisions in the gas cell being set such that each parent peptide ion is subject to only one hit and therefore only one cleavage event
This cleavage most frequently occurs at a peptide bond in the backbone of the parent peptide chain
So from each group of parent peptides of a selected mass, a “nested” set of fragments are produced
The mass difference between 2 adjacent fragments (daughter fragments) corresponds to the residue that differs between them
So the sequence of peptide can be determined by working along the ion series spelling out each amino-acid in the chain
This is done with the assistance of software but even with this, depending on the quality of the spectrum produced, can take significant amounts of user time
The resulting amino-acid sequence can then be used in a database search e.g. BLAST to try and identify the protein
The spectrum here shows the daughter ions produced from one parent peptide
in the nested set, the mass difference between 2 adjacent fragments (peptide ions/daughter fragments) corresponds to the residue that differs between the two fragments
So the sequence of peptide can be determined by working along the ion series spelling out each amino-acid in the chain.
The resulting amino-acid sequence can then be used in a database search e.g. BLAST to try and identify the protein
Even when the quality of the spectrum is poor, it is often possible to pick out clean peaks, and read off residues of sequence – 3 is enough, combined with other information (e.g. mass data of peptide and parent) to form a good search
the series of fragments corresponds to the sequence of the peptide

5. De novo sequencing
Sequence reads in N to C direction - PSGASTGVHEAMR

6. peptide de novo sequencing
tandem MS / de novo sequences used for
database searches for protein identification eg BLAST
confirm sequence of engineered mutations, alleles, etc
examine modifications –
biotinylation, acetylation, oxidation,
phosphorylation, glycosylation

7. Modifications e.g. N-terminus
Methionine removed
Serine acetylated
S-acetyl