Sample Preparation Enzymatic Digestion (Trypsin) + Fractionation

LC-MS: 1 MS spectrum / second Single Stage MS Mass Spectrometry LC-MS: 1 MS spectrum / second

Tandem MS Secondary Fragmentation Ionized parent peptide Tandem mass spectrometry selects one of the intense peaks observed in the single stage mass spectrum and further fragments all peptides with the selected mass to charge ratio. The tandem mass spectrum typically contains mass to charge ratio information about fragments of a a single peptide. Secondary Fragmentation Ionized parent peptide

Product Ion Scan Mode in a Triple Quadrupole Q1 Mass selection Q2 collision cell Q3 Full Scan A C B Ion C C+Ar Products Products D Ions in Source Q1 only transmits Ion C Fragment Ion C Q3 Scans for products

The peptide backbone The peptide backbone breaks to form fragments with characteristic masses. H...-HN-CH-CO-NH-CH-CO-NH-CH-CO-…OH Ri-1 Ri Ri+1 N-terminus C-terminus Tandem MS can be used to determine the amino-acid sequence of a peptide because proteins are made up of amino-acid chains. During secondary fragmentation, the peptide backbone breaks forming fragments with characteristic masses. AA residuei-1 AA residuei AA residuei+1

Ionization The peptide backbone breaks to form fragments with characteristic masses. H+ H...-HN-CH-CO-NH-CH-CO-NH-CH-CO-…OH Ri-1 Ri Ri+1 N-terminus C-terminus The parent peptide, when ionized, has at least one additional proton attached. AA residuei-1 AA residuei AA residuei+1 Ionized parent peptide

Fragment ion generation The peptide backbone breaks to form fragments with characteristic masses. H+ H...-HN-CH-CO NH-CH-CO-NH-CH-CO-…OH Ri-1 Ri Ri+1 N-terminus C-terminus When the peptide backbone breaks, the ionizing protons are retained on some of the fragments, which can then have their mass to charge ratio measured. Shown here is a suffix fragment, where the ionizing proton is retained on the C-terminus side of the backbone cleavage site. Also possible is a prefix fragment, where the ionizing proton is retained on the N-terminus side. AA residuei-1 AA residuei AA residuei+1 Ionized peptide fragment

Cleavages Observed in MS/MS of Peptides bi yn-i zn-i low energy ai xn-i vn-i wn-i -HN--CH--CO--NH--CH--CO--NH- Ri CH-R’ R” high energy ci di+1

Different MS-MS Instruments Yield Different Spectra A typical QTOF or triple quad MS-MS spectrum of a tryptic peptide contains a continuous series of y-type ions. The b-type ions are usually seen only at lower masses below the precursor m/z value Ion trap CID data of tryptic peptides is different in that one often finds a continuous series of both b-type and y-type ions throughout the spectrum

MS-MS for Protein ID Proteins are isolated (from gel or HPLC) and subjected to tryptic digestion Peptides are sent through ionizer and into a collision cell where the doubly charged ions are selected and fragmented through collision induced decay (CID) The resulting singly charged ions (daughter ions) are analyzed to determine the sequence or to ID the parent peptide

Why Trypsin for MS-MS? CID of peptides less than 2-3 kD is most reliable for MS-MS studies – The frequency of tryptic cleavage guarantees that most peptides will be of this size Trypsin cleaves on the C-terminal side of arginine and lysine. By putting the basic residues at the C-terminus, peptides fragment in a more predictable manner throughout the length of the peptide

Why Double Charges? Easiest spectra to interpret are those obtained from doubly-charged peptide precursors, where the resulting fragment ions are mostly singly-charged Doubly-charged precursors also fragment such that most of the peptide bonds break with comparable frequency, such that one is more likely to derive a complete sequence

How the peptide sequencing works? Use Tandem MS: two mass analyzer in series with a collision cell in between Collision cell: a region where the ions collide with a gas (He, Ne, Ar) resulting in fragmentation of the ion Fragmentation of the peptides in the collision cell occur in a predictable fashion, mainly at the peptide bonds (also phosphoester bonds) The resulting daughter ions have masses that are consistent with known molecular weights of dipeptides, tripeptides, tetrapeptides… Ser-Glu-Leu-Ile-Arg-Trp Collision Cell Ser-Glu-Leu-Ile-Arg Ser-Glu-Leu-Ile Ser-Glu-Leu Etc…

MS-MS & Peptide Fragments When peptides are proteins are admitted to a collision cell the peptide usually fragments at the weakest bond (the peptide bond, but some CH-NH and CH-CO breakage also occurs) Collision conditions have to be optimized for each peptide Two main types of daughter ions are produced -- “b” ions and “y” ions

MS-MS Peptide Fragmentation yn-1 yn-2 y1 R1 R2 R3 Rn H2N-CH-CO-NH-CH-CO-NH-CH-CO…CO-NH-CH-CO2H b1 b2 bn-1 b1 y1 b2 y2 b3 y3 b4 y4 b5 y5 signal

Peptide Fragmentation E=Glu G=Gly S=Ser F=Phe N=Asn P=Pro V=Val A=Ala R=Arg Peptide Fragmentation => E G S F F G E E N P N V A R 175.10 246.14 345.21 459.25 556.30 670.35 799.39 928.43 985.45 1132.52 1279.59 1366.62 1423.64 1552.69 = = =

MS-MS Peptide Fragmentation Ala-Gly-His-Leu-….Phe-Glu-Cys-Tyr b1 y1 b2 y2 b3 y3 b4 y4 b5 y5 signal

Peptide sequencing by mass spectrometry N-term. A B C D E C-term. Peptide molecules are fragmented by collisionally activated dissociation (CAD) collisions with neutral background gas molecules (nitrogen, argon, etc) typically dissociate by cleavage of -CO-NH- bond A B A B C D A A B C A B C D E m/z N-terminal product ions

Peptide Sequencing

Protocols for MS-MS Sequencing Usually can’t tell a “b” ion from a “y” ion Assume the lowest mass visible in the spectrum is a lysine or arginine (this is the y1 ion) this is because trypsin cuts after a lysine or arginine This y1 mass should be 147.113 for lysine or 175.119 for arginine {The y1 ion is calculated by adding 19.018 u (three hydrogens and one oxygen) to the residue masses of lysine and arginine}

MS-MS Sequencing Use the remaining “unassigned” peaks to see if you can construct a “b” ion series The highest mass peak corresponds to the parent ion or parent minus 147 (K) or 175 (R) The “b” ions give the “normal” sequence Both forward (b ion) and backward (y ion) sequences should be consistent Use the resulting sequence tag to search the databases using BLAST (remember to use a high Expect value ~ 100) to see if the sequence matches something

Peak Assignment 88 145 292 405 534 663 778 907 1020 1166 b ions S G F L E E D E L K 1166 1080 1022 875 762 633 504 389 260 147 y ions y6 100 Peak assignment implies Sequence (Residue tag) Reconstruction! y7 % Intensity [M+2H]2+ An assignment of the peptide fragments to the spectral peaks. You can see there is an excellent correspondence between the fragments that are expected and the observed spectral peaks. Note too the presence of the doubly charged, unfragmented parent ion. y5 b3 b4 y2 y3 y4 b5 y8 b6 b8 b9 b7 y9 250 500 750 1000 m/z

Peptide Sequencing by Mass Spectrometry LVDKVIGITNEEAISTAR Cysteine Synthase A 242 259 b3-17 y12 1261.4 100 y10 1091.5 y13 1374.5 Rel. Abund. b6 668.4 b8 b5 y9 838.5 y14 555.4 990.5 y11 y5 y4 b7 1474.4 b4 b9 b3 y6 y8 y7 b10 b11 b14 b12 b13 b15 b16 b17 400 600 800 1000 1200 1400 1600 1800 m/z

“+2” spectra

“bad” spectra

Modifications Some residues may be modified during the sample preparation procedure This introduces discrepancies in the expected and observed masses For example, Met residues are often oxidized

Mass of PTMs Mass Change Modification +14.0 Methylation   +14.0 Methylation +16.0 Hydroxylation +28.0 Formylation +30.0 Nitrosylation +42.0 Acetylation +80.0 Sulfation +80.1 Phosphorylation +180 Mono-glycosylation +204.4 Farnesylation +210.4 Myristoylation

MS/MS of Peptides Containing Phosphotyrosine 200 400 600 800 1000 1200 1400 1600 m/z 100 % K R Y 1167.59 1066.54 415.26 201.14 951.50 708.46 545.39 1687.85 1459.72 1441.68 D T E L G, G Yp y1 y3 y4 y5 y6 y7 y8 y9 y10 y11 y12 MH 163 243 Relative Intensity