Article Figures & Data
Figures
- Fig. 1.
Proof-of-concept: LC-MS/MS analysis of a dilution series of light/heavy (1:1 ratio) synthetic Pou5f1 reference peptide FEALQLSLK spiked into digested yeast extract. A, panel I, correlograms showing TCorr values obtained for the isotope-labeled (heavy) target peptide at different spike levels at the expected target retention time window of ±2 min (estimated based on the sentinel marker peptides as described in supplemental methods). The arrowheads (and solid lines) indicate the center of the estimated retention time, whereas the arrows (and dashed lines) show the observed retention time (apex of chromatographic peak recorded for the heavy peptide) if different from estimated time. A, panel II, extracted ion chromatograms of the major product ions (m/z 772.50 and 780.50) generated by the light (unlabeled) and heavy target peptides (precursor m/z 524.8055 and 528.8089, respectively). A, panel III, extracted ion chromatograms of the target precursor ions (m/z 524.8055 and 528.8089), respectively, of high resolution spectra (R = 60,000) obtained in Orbitrap. The star (*) indicates an isobaric interference (precursor m/z 524.7492 or 524.75, rounded). B, calibration curve showing AUC values calculated for extracted ion chromatograms of the product ion m/z 772.5 (target peptide precursor m/z 524.8055) obtained on an LTQ-Velos ion trap (squares) and the corresponding precursor ion (m/z 524.8055) signal acquired in high resolution spectrum on an Orbitrap (filled triangles). Inset, zoom-in. Arrows mark the LOD of 13.1 fmol and LOQ of 39.3 fmol, respectively. C, zoom-in (m/z 524.30–530.30) of a high resolution full scan spectrum obtained on an Orbitrap of a 100 fmol spike-in experiment showing the isotope envelopes of both the light (L) and heavy (H) precursor ion target and a dominant isobaric (m/z 524.7492) interference (I). Inset, distinct monoisotopic peaks of the light target and the interference. All shown retention time windows were estimated using linear regression of four retention time marker peptides (here called sentinels), which were monitored throughout the experiment. The expected retention time is the linear extrapolation of retention times from the sentinel position in each LC/MS/MS run (for linear regression see supplemental Fig. 2 and supplemental Table 2).
- Fig. 2.
Generation of a high quality reference MS/MS-spectral library. A, solubilized synthetic peptides are analyzed by electrospray ionization, with targeted isolation and fragmentation using an ion trap or triple quadrupole mass spectrometer and the product ions recorded continuously over 5 min. B, collected MS/MS spectra sequence-verified by database searching using SEQUEST. C, characteristic experimental intensity patterns of distinguishing b- and y-ion features stored in a relational database. D, MRM assay development using empirically optimized collision energy settings, shown here based on the breakdown curves obtained for the 10 most intense transitions (see “Experimental Procedures” and supplemental Methods” for details).
- Fig. 3.
Multiplex assay development. A, representative scheduled LC-MS/MS assay monitoring multiple reporter peptides corresponding to transcription factors regulating embryonic stem cell fate. Four exogenous sentinel marker peptides are jointly monitored in parallel to calculate relative retention time (stored in relational database) to control target data acquisition windows (gray highlights). The XIC of the most intense product ions of each peptide is indicated. B, heat map showing ion abundances for peptide standard curves generated using synthetic peptides alone or after spiking of the reference standards into a digested mESC NE. The zoom-ins show selectively linear signal response over 3 orders of magnitude (10-fold dilutions from 1 pmol to 1 fmol) using log-log (base 10) plots.
- Fig. 4.
Peptide identification and quantification using TCorr. A, representative correlograms showing the chromatographic profile of TCorr scores (0. 95 and higher) calculated for experimental MS/MS spectra recorded for spiked synthetic (left) or endogenous (right) Pou5f1 peptide FEALQLSLK in mESC NE matched against the corresponding reference spectral library over a specified retention time window 66- 86 min. Significant similarity in the observed b- and y-product ion intensity patterns occurs regardless of background interferences, allowing the XIC of the most intense target precursor-to-product ion transition to be reliably quantified. The gray highlights correspond to high confidence matches represented by the highest consecutive TCorr correlation scores. B, comparison of exemplar MS/MS spectra obtained for spiked and endogenous peptide (top) versus the corresponding library reference (bottom). XCorr correlation scores of a SEQUEST database search are indicated and obtained for the correct sequence FEALQLSLK. C, representative relative peak intensities of all b/y-ion transitions (TCorr-FULL) and TCorr-correlation score of 0.963 are shown. D, improved TCorr score 0.987 for endogenous peptide based on selected transitions freed from potential interferences (TCorr-TRANSITION).
- Fig. 5.
Multiplexed quantitative assays of pluripotency pathway components. A, heat map showing estimated protein abundance of Jak1, STAT3, STAT1, Akt1, and LIFR in cytoplasmic extracts from pluripotent mESC populations based on two endogenous or spiked peptides (1 and 2) measured by MRM. Inset, representative (untransformed) relative intensity (y axis). B, pathway schematic showing experimental XIC values (I, intensity) obtained for peptides corresponding to endogenous Sall4, Pou5f1, and Sox2 levels in nuclear and cytoplasmic extracts from +LIF (self-renewal; red) and −LIF (differentiation; green) treated mESCs. C, actual protein abundance measurements of pluripotency factors in +LIF and −LIF mESC NEs as obtained by targeted LC-MS/MS experiments (TPM) on an LTQ-ion trap.
Tables
- Table I
Selected proteins, accession numbers, protein class, and number of optimized peptides detected by LC-MRM-MS (a) and LC-TPM-MS (b)
Protein Swiss-Prot accession no. Protein classa Peptides predicted Assay optimization Successfully detected as endogenous a. LC-MRM Jak1 P52332 K 22 22 3 Stat1 P42225 TF 6 6 1 Stat3 P42227 TF 13 13 1 Lifr P42703 R 3 3 3 Akt1 P31750 K 15 13 5 P85A P26450 K 31 30 3 Total peptides 90 87 16 b. LC-TPM Pou5f1 P20263 TF 6 6 1 Sox2 P48432 TF 6 5 1 Klf4 Q60793 TF 1 1 0 c-Myc P01108 TF 3 3 0 Nanog Q80Z64 TF 1 1 0 Sall4 Q8BX22 TF 7 6 1 Total peptides 24 22 3 ↵a TF, transcription factor; R, receptor; K, kinase.
Supplemental Data
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