Top-down/Bottom-up Mass Spectrometry Workflow Using Dissolvable Polyacrylamide Gels

Abstract

Biologists' preeminent toolbox for separating, analyzing, and visualizing proteins is SDS-PAGE, yet recovering the proteins embedded in these polyacrylamide media as intact species is a long-standing challenge for mass spectrometry. In conventional workflows, protein mixtures from crude biological samples are electrophoretically separated at high-resolution within N,N'-methylene-bis-acrylamide cross-linked polyacrylamide gels to reduce sample complexity and facilitate sensitive characterization. However, low protein recoveries, especially for high molecular weight proteins, often hinder characterization by mass spectrometry. We describe a workflow for top-down/bottom-up mass spectrometric analyses of proteins in polyacrylamide slab gels using dissolvable, bis-acryloylcystamine-cross-linked polyacrylamide, enabling high-resolution protein separations while recovering intact proteins over a broad size range efficiently. The inferior electrophoretic resolution long associated with reducible gels has been overcome, as demonstrated by SDS-PAGE of crude tissue extracts. This workflow elutes intact proteins efficiently, supporting MS and MS/MS from proteins resolved on biologists' preferred separation platform.

Figure 1

Dissolvable polyacrylamide gels for high-resolution protein electrophoresis. (a) N,N′-cystamine-bis-acrylamide (BAC). (b) Schematic of the protein recovery from a BAC-crosslinked polyacrylamide gel. Reductively cleaving disulfide bonds in BAC is effective for releasing in-gel proteins. (c) Representative images of protein separation using BAC-PAGE. Protein extracts prepared from Drosophila tissues (20 μg total protein/lane) were separated on BAC gels and stained with Coomassie brilliant blue (CBB). (d) Reductive treatment of BAC gel pieces. An excised gel piece (1% TEMED concentration) containing a pre-stained protein band (Red: 75 kDa) was completely solubilized in 50 mM TCEP within 30 minutes.

Figure 2

Quick dissolution of BAC-crosslinked acrylamide gels. (a) Small gel pieces containing CBB-stained standard proteins (bovine serum albumin (BSA) and HIST1H3A) were dissolved in 90 μL of 50 mM TCEP for 10 min with a vigorous shake. (b) The dissolved gels were mixed with 30 μL of 4×NuPAGE LDS sample loading buffer and subjected to separation on 4–12% NuPAGE.

Figure 3

Top-down/bottom-up MS workflow using BAC-PAGE. (a) Analytical scheme for sample pretreatment using BAC-PAGE. (b) Evaluation of the recovery performance. A crude protein extract from Drosophila brain (10 μg total protein) was prepared by the procedure described in Figure 3a. Following methanol/chloroform precipitation, BAC gel-recovered proteins were revealed by Coomassie staining a 4–12% NuPAGE gel. *: whole tissue extract (5 μg total protein/lane). BAC: BAC-crosslinked 8% (w/v) acrylamide gel. (c) Top-down MS of intact HIST1H4A derived from BAC gel. Fragmentation of the (M + 13H)¹³⁺ molecule of human HIST1H4A was achieved by collision activated dissociation (CAD) and electron capture dissociation (ECD) in ESI FT-ICR mass spectrometer. Major CAD fragment ions are labeled and shown on the inset figure.

Figure 4

ESI-qTOF MS analysis of BSA from (a) BAC gel and (b) directly without BAC gel separation.

Figure 5

Comparative evaluation of different sample treatments for bottom-up proteomics. After tryptic digestion of a yeast protein extract (Promega) using different three methods independently, the digested peptides were subjected to LC-MS/MS analysis. For protein identification, MS/MS spectra were searched against the UniProt yeast proteome database by ProteinPilot using the following parameters: cys alkylation, iodoacetamide; digestion, trypsin; processing parameters, biological modification; and search effort, through ID. The values represent the averages from three independent experiments ± S.D.

Figure 6

ESI-FT-ICR mass spectrum from gel-separated Drosophila eye proteins.