Quantitative isoform-profiling of highly diversified recognition molecules
- University of Basel, Switzerland
Figures
Figure 1 with 7 supplements
Establishment and validation of SRM-assays.
(A) Proteotypic peptides for relative quantification of neurexin variants. The position of peptides within the overall domain structure of neurexin proteins is indicated (laminin-G and EGF-domains …
Figure 1—figure supplement 1
List of MS/MS spectra of proteotypic endogenous NRX1-pan and NRX1-alpha peptides, retention times and transition patterns of corresponding synthetic heavy peptides used for relative quantification in this study.
https://doi.org/10.7554/eLife.07794.004
Figure 1—figure supplement 2
List of MS/MS spectra of proteotypic endogenous NRX1-beta, NRX1-AS3(+), NRX1-AS6(+) and NRX1-AS4(+) peptides, retention times and transition patterns of corresponding synthetic heavy peptides used for relative quantification in this study.
https://doi.org/10.7554/eLife.07794.005
Figure 1—figure supplement 3
List of MS/MS spectra of proteotypic endogenous NRX2-pan peptides, retention times and transition patterns of corresponding synthetic heavy peptides used for relative quantification in this study.
https://doi.org/10.7554/eLife.07794.006
Figure 1—figure supplement 4
List of MS/MS spectra of proteotypic endogenous NRX2-alpha, NRX2-beta and NRX2-AS4(+) peptides, retention times and transition patterns of corresponding synthetic heavy peptides used for relative quantification in this study.
Note: one NRX2-alpha specific peptide (VNDGEWCHVDFQR) could not be identified by shotgun MS/MS. However, read-outs obtained with this peptide were very close to values obtained with two other …
Figure 1—figure supplement 5
List of MS/MS spectra of proteotypic endogenous NRX2-AS3(+), NRX3-pan and NRX3-alpha peptides, retention times and transition patterns of corresponding synthetic heavy peptides used for relative quantification in this study.
https://doi.org/10.7554/eLife.07794.008
Figure 1—figure supplement 6
List of MS/MS spectra of proteotypic endogenous NRX3-alpha, NRX3-beta, NRX3-AS3(+) and NRX3-AS4(+) peptides, retention times and transition patterns of corresponding synthetic heavy peptides used for relative quantification in this study.
https://doi.org/10.7554/eLife.07794.009
Figure 1—figure supplement 7
MS/MS spectrum of proteotypic endogenous NRX3-AS6(+) peptide, retention time and transition pattern of corresponding synthetic heavy peptide used for relative quantification in this study.
https://doi.org/10.7554/eLife.07794.010
Figure 2 with 2 supplements
Quantitative comparison of relative neurexin variant levels across brain areas.
(A, B) Relative amounts of total neurexin (NRX-pan), NRX-alpha, beta, and splice insertions across P30 mouse brain (OB = olfactory bulb, CX = cortex, HIP = hippocampus, TH = thalamus, ST = striatum, …
Figure 2—figure supplement 1
Preparation of synaptic proteins enriched Triton-Resistant-Membranes (TRM) from mouse brain.
(A) Workflow of the preparation of Triton X-100 resistant membranes (TRM) from brain tissues for enrichment of synaptic proteins. (B) Hierarchical clustering of log-transformed protein normalized …
Figure 2—figure supplement 2
Comparison of enrichments of neurexins and other synaptic proteins in TRM and Postsynaptic-Density (PSD) preparations.
(A) SRM based quantification of the enrichment of neurexin proteins in TRM and PSD samples (left diagram). . In both preparations comparable enrichment (∼4 fold) could be observed. No bias in …
Figure 3 with 1 supplement
Absolute quantification of endogenous neurexin isoforms.
(A) Western blot for recombinant proteins and SRM-based quantification approach using in vitro translated heavy amino acids labeled standards fused to GFP and as heavy standards in SRM assays. For …
Figure 3—figure supplement 1
Examples for the determination of lower limits of detection (LOD) and quantification (LOQ) for selected NRX1-3, PSD95 and N-CAD peptides used in this study for absolute quantification.
Peptide-specific standard curves were derived by spiking each digested synaptic protein-construct at a concentration range of five values into a digested mixture of mouse whole brain TRM protein …
Figure 4
Receptor recognition specificity profiling with splice site-specific SRM assays.
(A) Ratios of normalized spectral abundance factor (NSAF) of NRX1, 2, 3-specific peptides recovered from NL1B, NL3A2, LRRTM2-affinity matrices detected in shotgun experiments. Means of the …
Additional files
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Supplementary file 1
Lists of transitions and limits of detection and quantification for proteotypic peptides used in this study.
(A) Complete list of monitored transitions of peptides for neurexin and other proteins used in this study. (B) Complete list of Limits of detection (LOD) and quantification (LOQ) for peptides derived from in vitro expressed neurexin constructs used for absolute quantification of neurexin isoforms in this study.
- https://doi.org/10.7554/eLife.07794.017
