Parimal Samir Andrew J. Link Lab Vanderbilt University School of Medicine Nashville TN USA

1. Quantitation using Pseudo-Isobaric Tags (QuPIT) and Quantitation using Pseudo-isobaric Amino acids in Cell culture (QuPAC) Parimal Samir Andrew J. Link Lab Vanderbilt University School of Medicine Nashville TN USA

2. QuPIT and QuPAC Overview • We define pseudo isobaric as a set of chemically identical molecules that have the same integer mass but different exact masses depending upon the mass of the isotopes. • For Example, a hypothetical molecule CN will have the same interger mass of 27 amu if it has one C12 and one N15 atom or one C13 and one N14 atom. • The exact mass of the molecule with one C12 and one N15 atom is 27.000109 amu. • While the exact mass of the molecule with one C13 and one N14 atom is 27.006429 amu. • Therefore the mass difference between the molecules would be 0.00632 amu. • These two pseudo isobaric molecules can be distinguished in current high resolution mass spectrometers (Orbitrap and FT-ICR). Orbitrap Elite mass spectrometer would be used from here on as an example because they have enough resolution and are more cost effective than FT-ICR mass spectrometers. • A tag with 2 carbon and 2 nitrogen (2CN module) atoms can be designed to have a mass difference of 0.01264 amu. • This is a sufficient mass difference to separate peptides with m/z values up to 2000 with charge state 2 on Orbitrap Elite mass spectrometer. • A tag with 3 carbon and 3 nitrogen (3CN module) atoms can be designed to have a mass difference of 0.01896 amu. • This is sufficient mass difference to separate peptides with m/z values up to 2000 with charge state 3 on Orbitrap Elite mass spectrometer.

3. QuPIT and QuPAC Overview (continued...) • Since majority of tryptic peptides in LC-NSI-MS/MS experiments have +2 or +3 charge, A tag with 3 carbon and 3 nitrogen atom would be enough to distinguish the differentially labeled peptides on Orbitrap Elite mass spectrometer. • Furthermore, vast majority of tryptic peptides identified in a LC-NSI-MS/MS experiment have m/z less than 1200. • Therefore a tag with only 2 carbon and 2 nitrogen atoms would be able to quantitate most of the peptides in the complex mixture derived from tryptic digestion on Orbitrap Elite mass spectrometer. • Similarly, pseudo isobaric amino acids can be designed that can be added to cell culture media for metabolic labeling of proteins. • For a workflow involving trypsin digestion, pseudo isobaric lysine and arginine can be designed because • trypsin cleaves C-terminal to lysine and arginine. • Also, they have at least 2 carbon and nitrogen atoms each.

4. QuPIT Overview • Chemical tagging based approach • Tag can be attached to primary amines using NHS esters. • Alternatively, -SH, -OH or C00H reactive tags can also be used, depending upon which amino acid needs to be tagged. • Tags will have mass difference of < 0.1 Da. • This will allow simultaneous isolation and fragmentation of peptides from all the labeled samples. • Tags differing only in stable isotopes, therefore they should behave similarly during chromatography. • Quantitation using the precursor ion isotopic envelope. • Will require ultra high resolution MS instruments. Tag 1 Tag 2

5. QuPAC Overview • Cells to be compared are grown in the presence of one type of pseudo isobaric amino acids or other • Sample 1 was grown with C13N14 arginine and lysine • Sample 2 was grown with C12N15 arginine and lysine • Cells are lysed and lysates are mixed together • The mixture of lysates is processed and analyzed by LS-MS/MS • Peptides are quantitated by integrating the peak areas of precursor ions. Sample 1 Sample 2 QuPAC

6. Comparison QuPIT with Other Precursor Ion Based Approaches QuPIT mTRAQ, ICAT, SILAC • At least two fold reduced sampling because pseudo-isobaric precursors are coisolated for fragmentation in tandem MS • Simpler alignment of isotopic envelopes. • Can be used with samples from any source. No need to label whole organisms. • Using 2CN or 3CN modules upto 7 or 5 samples can be multiplexed and the difference in masses of singly charged precursor would still be less than 0.1 FWHM. • At least two MS2 spectra per peptide (oversampling). • Difficulty with aligning isotope envelopes. • Can be used only with samples in cell culture. Labeling whole organisms prohibitively costly and time consuming. • At most 3 samples can be multiplexed

7. Comparison of QuPAC with Other Precursor Ion Based Approaches QuPAC mTRAQ, ICAT, SILAC • At least two fold reduced sampling because pseudo-isobaric precursors are coisolated for fragmentation in tandem MS • Simpler alignment of isotopic envelopes. • At least two MS2 spectra per peptide (oversampling). • Difficulty with aligning isotope envelopes.

8. Comparison Between iTRAQ/TMT and QuPIT iTRAQ/TMT QuPIT • Requires HCD fragmentation • Therefore cannot perform simultaneous precursor ion scan and fragment scan on hybrid instruments • Smaller number of MS2 spectra acquired • Signal suppression of reporter ions because of competing requirements for peptide identification and Quantitation • Quantitation with fragment ions, which is less sensitive compared to quantitation at precursor ion level • Quantitation affected by interference by coeluting peptides • Either CID or HCD fragmentation • Therefore can perform simultaneous precursor ion scan and fragment scan on hybrid instruments • A larger number of MS2 spectra acquired if using CID • No signal suppression because quantitation will be independent of peptide identification • Quantitation will be at precursor ion level, which is inherently more sensitive than quantitation with fragment ions • No interference by coeluting in quantitation, although peptide ID may be affected.

9. Summary • QuPIT and QuPAC combine the strengths of existing technologies to achieve maximum sensitivity and precision in mass spectrometry based quantitation of small molecules, peptides and proteins. • Using the difference between integer mass and exact mass of isotopes can be used to design molecules that can be used for mass spectrometry based precise quantitation.