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Tandem Mass Spec (word-for-word lecture notes)

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Biological Sciences
Rongmin Zhao

Lec 11 BIOC23Slide 1Last week talked about general principle of mass spectrometryDescribed 2 applications for mass speco1 Direct determination of mass of a molecule including small like 300 daltons and large macromocs like proteins BSA66 kDa DETERMINE MOLECULAR WEIGHTo2 Mass spec fingerprinting after trypsin digestion can detect set of peptides in mass spec and search database to determine what exactly the protein isDETERMINE WHAT PROTEINo3 Tandem mass spec to determine peptide sequence tandem means 2 mass analyzersfiltersdetectors used side by side and pick one peptide and then determine peptide sequenceDETERMINE PEPTIDE SEQUENCESlide 2 Tandem Mass Spec Instrument How Peptide sequence WorksInstrument type hybrid orthogonal qTOF Q StaroTOFtime of flight mass analyzerAt the beginning you have ion source that will put charge on the mocsoThen ions pass thru first mass analyzer this first one is used to choose a peptide with a particular masscharge ratiooThen when peptide is selected it is passed to the next compartment called collision the particular peptide in collision cell can be fragmented by high pressure gas helium gas can break down peptide into smaller fragmentsoThen these smaller fragments are passed thru the next mass of analyzerTOF time of flight mass analyzer which analyzes the masscharge ratio of the fragmented peptide This is a diagram of how tandem mass spec worksAt the beginning you have a lot of peptides due to trypsin digestion and when pass this mix thru the first analyzer it will allow you to pick just one peptide then can fragment this chosen peptide and analyze and determine the sequenceSlide 3 Peptide fragmentation How collision cell works high pressure helium gas will be applied to peptide which will break down the peptide at particular sitesoPeptide bond is very fragile usually broken down by high pressure gas of collision celloHowever this breakage can be random slide 4Slide 4From the first mass analyzer choose a particular peptide as the one shown with 10 AA after you pass this peptide thru the collision cell it will be randomly fragmented by breaking the peptide bondgenerate mixture of randomly fragmented peptides
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