Enzymatic Digestion of Proteins in Gels for Mass Spectrometric Identification and Structural Analysis

Abstract

Enzymatic digestion of proteins is a key technique used in protein identification. By combining the digestion with mass spectrometric detection, proteins at very low femtomole levels, and in some cases subfemtomole levels, can be identified. Typically, one- or two-dimensional SDS-PAGE is used to isolate the proteins of interest, followed by staining with Coomassie blue, digestion-compatible silver stain, or Sypro Ruby for detection. Two-dimensional (2-D) fluorescence difference gel electrophoresis (DIGE), which uses Cy3 and Cy5 dyes for detection, allows comparison of two different sample states in order to locate proteins that are up- or down-regulated. In each case, an in-gel digestion, usually tryptic, is used with mass spectrometry to identify these proteins of interest. For large numbers of gel spots, robotic digestion can save time and money.

A tutorial on the use of temporal principal component analysis in developmental ERP research – Opportunities and challenges

“…Whereas measurement invariance of the topography is clearly plausible in many situations ( Delorme et al, 2012 ), spatial PCA requires the use of special rotation techniques because factors typically contribute to the voltage at all electrode sites. These rotation techniques (e.g., Infomax ) are closely related to independent component analysis (ICA; Dien et al, 2007 ), and they aim to extract factors which are as stochastically independent as possible instead of rotating towards a simple structure (for mathematical introductions and tutorial treatments, see, e.g., De Lathauwer et al, 2000 ; Groppe et al, 2008 ; Huster and Raud, 2018 ; Makeig et al, 1996 ; Stone, 2004 ). Please note that there is an ongoing debate regarding the use of restricted versus unrestricted solutions for spatial decompositions as well ( Artoni et al, 2018 ).…”

Selective Proteomic Proximity Labeling Assay Using Tyramide (SPPLAT): A Quantitative Method for the Proteomic Analysis of Localized Membrane‐Bound Protein Clusters

“…Liquid chromatography system (e.g., nanoAcquity UPLC system, Waters Corp.) Tandem mass spectrometer (e.g., LTQ Orbitrap Velos hybrid ion trap mass spectrometer, Thermo Scientific) Cover slips Confocal microscope Additional reagents and equipment for determination of protein concentration (UNIT 3.4; Olson and Markwell, 2007), one-dimensional SDS gel electrophoresis of proteins (UNIT 10.1; Gallagher, 2012), staining proteins in gels (UNIT 10.5; Echan and Speicher, 2002), immunoblot detection (UNIT 10.10; Gallagher, 2001), and in-gel digestion of proteins (UNIT 11.3; Stone and Williams, 2004) 1. Remove cryosections from −80°C, and immediately block for 1 hr at 4°C in PBS containing 1% BSA, 0.1% saponin, and protease inhibitors to permeabilize any membranes.…”

“…Additional reagents and equipment for determination of protein concentration (UNIT 3.4; Olson and Markwell, 2007), one-dimensional SDS gel electrophoresis of proteins (UNIT 10.1; Gallagher, 2012), staining proteins in gels (UNIT 10.5; Echan and Speicher, 2002), immunoblot detection (UNIT 10.10; Gallagher, 2001), and in-gel digestion of proteins (UNIT 11.3; Stone and Williams, 2004) Culture cells 1. Seed DT40 cells (day 0) at 1 × 10 5 cells/ml in RPMI supplemented with 10% FBS, 1% chicken serum, 100 U/ml penicillin, and 100 μg/ml streptomycin, and incubate at 37°C, 5% CO 2 .…”

Selective Proteomic Proximity Labeling Assay Using Tyramide (SPPLAT): A Quantitative Method for the Proteomic Analysis of Localized Membrane‐Bound Protein Clusters

“…Laboratories), dialyzed 10% FBS (Dundee Cell Products), and dialyzed 1% chicken serum (Sigma) RPMI 1640 SILAC light medium containing non-labeled L-lysine and L-arginine (K 0 R 0 ) supplemented with 2 mM L-glutamine (PAA Laboratories), dialyzed 10% FBS (Dundee Cell Products), and dialyzed 1% chicken serum (Sigma) Fe 3+ -loaded, HRP-conjugated apotransferrin (see Support Olson and Markwell, 2007), one-dimensional SDS gel electrophoresis of proteins (UNIT 10.1; Gallagher, 2012), staining proteins in gels (UNIT 10.5; Echan and Speicher, 2002), immunoblot detection (UNIT 10.10; Gallagher, 2001), and in-gel digestion of proteins (UNIT 11.3; Stone and Williams, 2004) 1. On day 0, seed DT40 cells at 1 × 10 5 cells/ml in RPMI supplemented with 10% FBS, 1% chicken serum, 100 U/ml penicillin, and 100 μg/ml streptomycin.…”