1. Quantification of low-abundance proteins in complexes and in total cell lysates by mass spectrometry Bastienne Jaccard and Manfredo Quadroni Université de Lausanne Faculté de biologie et de médecine Department of Biochemistry RESULTS SDS-PAGE of the sample (immunoprecipitation or total cell lysate) followed by proteolytic digestion with trypsin (cleaves after lysine or arginine residues) Fractionation of the complex mixture of tryptic peptides by liquid chromatography. The chromatography unit is directly coupled to the mass spectrometer. Sample analysis in the MRM scan mode in a mass spectrometer (triple quadrupole) Q1 = First quadrupole DetectorIon source Q1 is set to transmit only the wanted m/z (mass to charge ratio), the m/z of one tryptic peptide belonging to the protein we want to quantify Q2 = Second quadrupole The selected tryptic peptide is fragmented in fragment ions in Q2 Q3 = Third quadrupole Q3 is set to transmit only the wanted m/z, the m/z of one fragment ion derived from fragmentation of the selected tryptic peptide Elution time Intensity (cps) Signal for the transition parent ion (tryptic peptide)  fragment ion METHOD Detection of a low-abundance protein (FADD) in a total cell lysate 510152025303540455055 Time, min 0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 Intensity, cps 510152025303540455055 Time, min 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500 Intensity, cps Analysis of an immunoprecipitation sample (Fas Death-Induced Signaling Complex or DISC) by MRM MRM of BAFF/FasL pulldown 0 5000 10000 15000 20000 25000 30000 35000 40000 FasL Fas Casp-8 Casp-10 BAFF BAFFR Ro52 Ef-1-1 Tubulin PIMT 14-3-3b GAPDH protein cps (surface) BAFF FasL Flip Fadd Ran Proteins of the DISC Contaminants Ef-1-1 Ro52 Ef-1-1 Ro52 Caspase-8 FasL Fas Caspase-8 Flip Negative control : immunoprecipitation of BAFF Analysis by MRM Immunoprecipitation of the Fas DISC Analysis by MRM Several transitions were monitored for proteins known to be in the Fas DISC (Fas, FasL, FADD, caspase-8, caspase-10, Flip), for proteins known to be only in the negative control (BAFF, BAFFR) and for contaminants (EF-1-1, Ro52, Ran, GAPDH, tubulin, PIMT). In figure 1 and 2, we can see two MRM plots (Fas DISC and negative control). Several transitions were monitored in the same run. The same transitions were monitored for both samples. In figure 3 is represented the intensity of the signal (peak area) obtained in both samples for each transition. A signal was detected for contaminants in both samples but this was not the case for proteins known to be specific to one sample tubulin 14-3-3 PIMT 14-3-3 PIMT BAFF tubulin 67.067.568.068.569.069.570.070.571.071.5 Time, min 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 168 Intensity, cps 68.69 67.267.467.667.868.068.268.468.668.869.069.2 Time, min 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 Intensity, cps 68.04 The peptide ENATVAHLVGALR from FADD (m/z=676 if doubly charged and 451 if triply charged) was used to perform MRM. In figure 4, we see the fragmentation pattern of this tryptic peptide. The fragmentation pattern is reproducible and the most intense fragment ions were used to set the transitions to monitor (figure 5 and figure 6). 836.51 765.47 628.41 515.33 m/z of fragment ions selected for MRM Transitions A 676  836.5 B 676  765.5 C 451  515.4 D 451  628.4 451=triply charged tryptic peptide 676=doubly charged tryptic peptide MRM of recombinant FADD MRM of total cell lysate Transitions A 676  836.5 B 676  765.5 C 451  515.4 D 451  628.4 451=triply charged tryptic peptide 676=doubly charged tryptic peptide INTRODUCTION Quantification of proteins can be performed in some mass spectrometers by using a particular method called MRM (multiple reaction monitoring). As we aim to quantify low-abundance proteins (proteins of the Fas Death-Induced Signaling Complex or DISC), it is crucial to determine if they can be relatively well detected by MRM. Here we show preliminary results that indicate that the low-abundance proteins we are working with are relatively well detected by MRM in an immunoprecipitated complex but also, for some of them, in a total cell lysate. CONCLUSION We are able to detect relatively well by MRM the low-abundance proteins that we aim to quantify. The higher the intensity of signal is, the better it is for quantification. To increase the intensity of signal, several parameters were optimized like the choice of the parent ion (tryptic peptide) and of the transition as well as the MRM parameters. A relatively good detection of FADD in total cell lysates was only possible after optimization. Now, the next step will be quantification. Absolute or relative quantification can be performed. Absolute quantification could allow us to determine the stoichiometry of the DISC or the number of copies of one protein in a particular cell line for example. Absolute quantification is performed by using an internal standard (which mimicks a tryptic peptide but is synthesized with a stable heavy isotope of one amino acid) put in the sample at a known concentration. Relative quantification could allow us to detect changes in the composition of the DISC or in the level of expression of some proteins between two conditions for example. Relative quantification is performed by differentially modifying (with light and heavy acrylamide for example) the proteins or peptides in the two conditions. Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PS For a given tryptic peptide, the intensity of signal depends on the chosen transition. Transition A gives higher signals than transition D.