Tissue Proteomics by One-Dimensional Gel Electrophoresis Combined with Label-Free Protein Quantification

Abstract

Label-free methods streamline quantitative proteomics of tissues by alleviating the need for metabolic labeling of proteins with stable isotopes. Here we detail and implement solutions to common problems in label-free data processing geared toward tissue proteomics by one-dimensional gel electrophoresis followed by liquid chromatography tandem mass spectrometry (geLC MS/MS). Our quantification pipeline showed high levels of performance in terms of duplicate reproducibility, linear dynamic range, and number of proteins identified and quantified. When applied to the liver of an adenomatous polyposis coli (APC) knockout mouse, we demonstrated an 8-fold increase in the number of statistically significant changing proteins compared to alternative approaches, including many more previously unidentified hydrophobic proteins. Better proteome coverage and quantification accuracy revealed molecular details of the perturbed energy metabolism.

Identification of a novel putative interaction partner of the nucleoporin ALADIN

“…Gel analyses were performed at the Mass Spectrometry Facility at the Max Planck Institute for Molecular Cell Biology and Genetics (Dresden) on a nano high-performance liquid chromatograph (UltiMate) interfaced on-line to a LTQ Orbitrap Velos hybrid tandem mass spectrometer as described previously (Vasilj et al, 2012). …”

The phosphatase Pgam5 antagonizes Wnt/β-Catenin signaling in embryonic anterior-posterior axis patterning

“…Eluates were digested with trypsin and subsequently with Lys-C in solution. After addition of protein digests as quantitative standards, the samples were desalted on C-18 stage tips (Nest Group) and analyzed by nanoflow HPLC-MS/MS (2D-NanoLC; Orbitrap Velos MS, Thermo Fisher Scientific; Vasilj et al, 2012). Protein identification was carried out using Mascot V2.2 (Matrixscience) and the determination of peptide ion signals for label-free quantification was performed using Progenesis LCMS V2.6 (Nonlinear Dynamics) and relative quantification was based on the most intense three signals (MI3) (Groessl et al, 2012;Silva et al, 2006).…”

Morphine Biosynthesis in Opium Poppy Involves Two Cell Types: Sieve Elements and Laticifers

“…SDS-PAGE of whole-stem and latex protein extracts from the opium poppy chemotype Roxanne contrasted the complexity of each subproteome and revealed the abundance of low molecular mass MLPs in laticifers (Figure 4). Using a gel-based liquid chromatography-tandem mass spectrometry approach (Schirle et al, 2003;Vasilj et al, 2012), 1518 and 511 distinct protein families were identified from the whole-stem and latex samples, respectively. Identification was based on searches of all available plant entries in the National Center for Biotechnology Information nonredundant (NCBInr) database.…”