Protein/Peptide Quantification An Introduction to Quantification by Isotope dilution Chemical labelling Biological labelling Advanced scanning methods

General SILAC Workflow

Quantification by Isotope Comparison

Complications are possible

Phosphopeptide analysis in the mitotic cell cycle

Complications are possible

Dynamic SILAC

Relative Isotope Abundance The variance is shown for two proteins The number of peptides vary but not the ratio

Decay curve fitting The curves all appear to fit a single exponential

Variation of decay Wide variation in decay rates: But why?

Multiplexing SILAC Label with 12C4, 13C4, 13C6, 13C6N4, 13C615N42H7

D does not affect elution

Western blot support

Chemical Labelling: iTRAQ 9.0 436.2 863.4 1290.6 1717.8 2145.0 20 114 115 116 117 Mass (m/z) 40 60 80 100 Isobaric Tag (Total mass 145 Da) Reporter (Mass 114 - 117) Balance (Mass 31 - 28) Peptide Reactive Group Charged Neutral Loss Amine Specific

iTRAQ labelling

iTRAQ reporter ions

iTRAQ 8-plex analysis

TMT - Isobaric Mass Tags Tandem Mass Tags A family of amine reactive isobaric MS/MS tags based on an identical chemical structure Six-plex profiling Tandem Mass Tags (TMT) are an intelligent way to obtain dramatically improved quantitation data allowing for peptide andprotein biomarker discovery, validation, and the development of multiplex assays for routine measurement with analytical performance equivalent to ELISA. Tandem Mass Tags belong to the family of reagents known as isobaric mass tags. As the term suggests, several different forms of a molecule have the same mass and can be independently attached to a peptide or protein via a reactive group. Two core elements the Mass Reporter (Tag) and the Mass Normaliser(MN) are connected via a Cleavable Linker, which has the tendency to break preferentially during MS/MS fragmentation conditions. By selective placement of isotopes such as 13C, 15N or 18O at certain positions of the Tagand the Mass Normaliser, the mass of the Tag can be varied to produce series of Mass Reporters, typically in 1 Dalton increments, while keeping the mass of the entire label constant.

Peptide identification and quantification Peptide sequence information Slide courtesy of Atul Deshpande and Travis LaFavor at Pierce

General TMT Workflow

AQUA - Absolute Quantitation of Peptides Must use internal standard Isotopically stable heavy peptides Rules for selection of a suitable (proteotypic) peptide Unique to the protein to be quantified Previously detected by mass spectrometry Preferred charge state 2+ Avoid peptides with Cys, Met residues Phosphopeptides can be used for monitoring phosphorylation events

AQUA Peptide Workflow Identify a proteotypic peptide Leu 13C6 Synthesize a heavy peptide Spiking and LC/MS analysis Quantitation from full scan MS

How a Mass Spectrometer works....... The ion source is the part of the mass spectrometer that ionizes the material under analysis (the analyte). The ions are then transported by magnetic and electric fields into the mass spectrometer. Ionisation should be a gentle as possible to ensure that an intact molecule is introduced into the mass spectrometer with the minimum of fragmentation. If a molecule is not charged, it cannot be directed by the magnetic or electric fields and will not reach the detector and will therefore not be seen.

Triple Quadrupole Scanning Modes All peptides measured intact Q1 Q2 Q3 m/z RIC

Product ion Scanning Mode (MS/MS) Only Green is selected for fragmentation Q1 Q2 Q3 m/z RIC

Parent Ion Scanning Mode All peptides sequentially fragmented Only parents of selected fragment detected Q1 Q2 Q3 m/z RIC

Searching for PhosphoPeptides

Parent ion triggered MS/MS

Selective GlycoPeptide Detection (HexNAc)

Single Reaction Monitoring Only peptides with chosen mass fragmented Only parents of selected fragment detected Q1 Q2 Q3 m/z RIC

Multiple Reaction Monitoring Only peptides with chosen mass fragmented Only parents of both selected fragments detected Q1 Q2 Q3 m/z RIC

MS/MS spectrum of the peptide LDVDQALDR

TIC of 16 MRM transitions for peptides 1-4 Endogenous and Isotopically labeled pairs

Replacing Western Blotting? Mutant Gene Name Western MRM heavy/light

Conclusions Proteomics is slowly becoming useful Up to 4000 proteins can be identified in a cell Dynamic range is around 100 and still limited MRM increases dynamic range to ca 5000 Quantification becoming possible but problematic