BIF401 Merged Slides 126-250

Lecture 126

MS1 & Intact Protein Mass

• Once a protein is ionized, it can be deflected by a magnetic field in proportion to its mass
• Proteins can then be measured for their mass in a Mass Spectrometer(MS).

Processing MS1:

• Mass/Charge ratio helps calculate the mass of the protein
• What if a protein carries multiple charges?
• “Mass-select” can help select a specific MS1 for further analysis.

Scoring:

• We can score each protein hit in a way that the better match gets a higher score while the low quality match gets a lower score • But how to be sure that a peak is from a multiple charged ion? • So once we have filtered away multiple charged peaks, we retain only those peaks with charge 1 • Next, we filter protein database and obtain candidate hits

with theoretical mass of proteins in the database

between experimental and theoretical mass

Resolving Multiple Charges

• MS1 spectra reports the intact protein masses for all proteins in a protein complex MScore = √
• Incase the sample contains peptides, MS1 gives the intact mass of the peptides Example: A Protein Sequence from Database “MQLF”

MW: 537.67 · Lecture 127 · Scoring Intact Protein Mass

• MS1 helps obtain the intact mass of the precursors molecules
• Precursor molecules may be proteins or peptides depending upon the proteomics protocol employed
• Using the masses reported by MS1, protein databases can be filtered for candidate protein matches (or hits)
• Remember to convert the masses of all molecules to 1+mass

Lecture 128

Protein Fragmentation Techniques

• MS1 helps measure the molecular weight of intact proteins or peptides • One can compare this mass with the theoretically calculated protein mass from database & score/rank matches.

• What about the case that several proteins obtain the same matching score?

• In this case, an other step may be required to further classify/score proteins in order to obtain the most probable candidate!

• What if we could fragment the ionized intact protein or peptide within the MS chamber?
• This would help measure the fragment masses in the same way as their precursors!

Tandem MS · Lecture 129 · Modifying MS1 to include fragmentation

• Intact proteins or peptides can be measured for their intact mass • This can be followed up by their fragmentation within the MS chamber • Can we measure the mass of these fragments?

MS2 and MS’n’:

• Tandem MS can be extended to the fragments of the intact fragment! • All you need is the MS instrument capability to (i) select fragment’s mass range (ii) fragment the precursor fragment

Techniques:

• Electron Capture Dissociation (ECD)
• Electron Transfer Dissociation (ETD)
• Collision Induced Dissociated (CID)

Fragmentation Products:

• Each fragmentation technique results in a specific type of fragment
• ECD gives out ‘C’ and ‘Z’ ions
• CID gives out ‘B’ and ‘Y’ ions etc..

Conclusions: If we can:

1. measure the mass of fragments using MS
2. Calculate the theoretical mass of the fragments

• Then, we can award score on the basis of the similarity of experimental and theoretical mass • Tandem MS helps in measurement of mass to the fragments as well! • This process provides an other step in further scoring and ranking • Protein identification thus becomes easier.

Lecture 130

Measuring Experimental Fragment’s Mass:

• The intact sample molecule may be fragmented into 2 afterward • The two fragments are measured by Tandem MS or MS2

• Intact molecule’s fragmentation splits the intact molecule into two parts • ECD _>c & z ions

Fragment’s Mass:

• Masses of fragments produced by MS2 depend upon the fragmentation technique used
• Because each fragmentation method splits the protein/peptide at a unique location

DB)

Lecture 131

Calculating Theoretical Fragment’s Mass:

• We compare these fragment masses with the theoretical fragments of proteins from sequence database.
• Depends upon the fragmentation technique, mass of the two theoretical fragments can be calculated.