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

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 amu. While the exact mass of the molecule with one C13 and one N14 atom is amu. Therefore the mass difference between the molecules would be 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 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 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.

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 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.

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

QuPAC Overview Cells to be compared are grown in the presence of one type of pseudo isobaric amino acids or other o Sample 1 was grown with C13N14 arginine and lysine o 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

Comparison QuPIT with Other Precursor Ion Based Approaches QuPIT 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. mTRAQ, ICAT, SILAC 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

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

Comparison Between iTRAQ/TMT and QuPIT iTRAQ/TMT 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 QuPIT 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 i nterference by coeluting in quantitation, although peptide ID may be affected.

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.