Transcript Slide 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 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. METHOD Sample analysis in the MRM scan mode in a mass spectrometer (triple quadrupole) SDS-PAGE of the sample (immunoprecipitation or total cell lysate) followed by proteolytic digestion with trypsin (cleaves after lysine or arginine residues) Ion source Detector Q1 = First quadrupole Q2 = Second quadrupole 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 Q3 = Third quadrupole The selected tryptic peptide is fragmented in fragment ions in Q2 Signal for the transition parent ion (tryptic peptide) fragment ion 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 Intensity (cps) Fractionation of the complex mixture of tryptic peptides by liquid chromatography. The chromatography unit is directly coupled to the mass spectrometer. Elution time RESULTS Ro52 Analysis of an immunoprecipitation sample (Fas Death-Induced Signaling Complex or DISC) by MRM 7500 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). Figure 1 7000 6500 6000 Analysis by MRM 5500 5000 4500 4000 3000 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 Caspase-8 2000 Fas Flip Ef-1-1 PIMT 14-3-3 2500 1500 1000 500 0 5 10 15 20 25 950 MRM of BAFF/FasL pulldown Contaminants 850 800 35000 750 45 50 55 Negative control : immunoprecipitation of BAFF Analysis by MRM 700 30000 650 25000 600 Intensity, cps 500 450 400 10000 tubulin BAFF 350 5000 300 0 Ran GAPDH 14-3-3b PIMT Tubulin Ro52 Ef-1-1 BAFFR BAFF Fadd Flip Flip Casp-10 Casp-8 Casp-8 Fas Fas FasL FasL 250 200 150 100 protein 50 0 5 Detection of a low-abundance protein (FADD) in a total cell lysate 10 68.04 3800 3400 B 676 765.5 C 451 515.4 D 451 628.4 2600 m/z of fragment ions selected for MRM 2400 451=triply charged tryptic peptide 2200 2000 676=doubly charged tryptic peptide 1800 1600 628.41 1400 1200 1000 800 600 200 0 67.2 67.4 67.6 67.8 68.0 68.2 68.4 Time, min 40 45 50 55 Transitions Transitions A 676 836.5 3000 35 MRM of total cell lysate 400 CONCLUSION 30 Time, min 68.6 68.8 69.0 69.2 Intensity, cps 836.51 25 Figure 6 3600 3200 Intensity, cps 765.47 20 MRM of recombinant FADD 2800 515.33 15 Figure 5 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). Figure 4 tubulin 15000 550 14-3-3 BAFF FasL 20000 PIMT cps (surface) 40 Ef-1-1 40000 Figure 2 900 35 Ro52 Figure 3 30 Time, min Caspase-8 3500 tubulin FasL Intensity, cps 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. Proteins of the DISC Immunoprecipitation of the Fas DISC 68.69 A 676 836.5 168 160 B 676 765.5 150 C 451 515.4 140 D 451 628.4 130 120 110 451=triply charged tryptic 100 peptide 90 676=doubly charged tryptic 80 peptide 70 60 50 40 30 20 10 0 67.0 67.5 68.0 68.5 69.0 69.5 70.0 70.5 71.0 71.5 Time, min PS For a given tryptic peptide, the intensity of signal depends on the chosen transition. Transition A gives higher signals than transition D. 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.