Journal: bioRxiv
Article Title: High-throughput proteomic and phosphoproteomic analysis of formalin-fixed paraffin-embedded tissue
doi: 10.1101/2024.11.17.624038
Figure Lengend Snippet: A. Proteome depth from 1 µg of Jurkat peptides analyzed using optimized DIA methods on the timsTOF HT and Exploris 480. Bars represent the average of (n=2 injections), with error bars showing the standard deviation. The Bruker timsTOF HT, equipped with a dual trapped ion mobility spectrometry (TIMS) tunnels, allows for parallel accumulation-serial fragmentation (PASEF), separating ions by their collisional cross-section (CCS), allowing precursor selection based on their ion mobility (IM) and mass to charge (m/z). Variable-window diaPASEF was optimized for FFPE proteomics using Jurkat peptide digests across four gradients (23, 30, 35, and 55 minutes) on a 25 cm PepSep column. The results were compared to data acquired on the Orbitrap Exploris 480 with 110-minute and 45-minute gradients using a 25 cm home-packed Reprosil C18 column. The wide-window DIA method on the Orbitrap Exploris480 utilized variable isolation windows ranging from 12 m/z to 24 m/z, adjusting to precursor density. Both methods aimed for six data points per peak (DPPP) for quantitative reproducibility. The timsTOF HT 35-minute gradient achieved ∼8,000 unique proteins, comparable to the 110-minute Orbitrap gradient, with a balanced unique peptide depth (∼135,000). B. Peptide identifications across the four methods tested. C. Four blocks from genetically engineered mouse models (GEMM) were used in the mouse FFPE experiments. Two blocks with total body Ncoa4 overexpression (OE) were compared to wild-type (WT) blocks. Blocks WT2 and OE2 included all organs, while WT1 lacked a few organs, and the OE1 block was missing the pancreas. D. Slide representation showing all organs embedded into the GEMM FFPE blocks.
Article Snippet: For Astral DIA (4 m/z), peptides were separated using a Vanquish Neo TM UHPLC system (Thermo Fisher) coupled to a Orbitrap Astral TM (Thermo Fisher) with a Aurora Elite TM TS C18 UHPLC column (IonOpticks, 15 cm length/75 µm inner diameter/1.7 µm particle size) at a flow rate of 800 nL/min.
Techniques: Standard Deviation, Ion-Mobility Spectrometry, Selection, Isolation, Over Expression, Blocking Assay
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