A robust two-dimensional separation for top-down tandem mass spectrometry of the low-mass proteome

Abstract

For fractionation of intact proteins by molecular weight (MW), a sharply improved two-dimensional (2D) separation is presented to drive reproducible and robust fractionation before top-down mass spectrometry of complex mixtures. The "GELFrEE" (i.e., gel-eluted liquid fraction entrapment electrophoresis) approach is implemented by use of Tris-glycine and Tris-tricine gel systems applied to human cytosolic and nuclear extracts from HeLa S3 cells, to achieve a MW-based fractionation of proteins from 5 to >100 kDa in 1 h. For top-down tandem mass spectroscopy (MS/MS) of the low-mass proteome (5-25 kDa), between 5 and 8 gel-elution (GE) fractions are sampled by nanocapillary-LC-MS/MS with 12 or 14.5 tesla Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. Single injections give about 40 detectable proteins, about half of which yield automated ProSight identifications. Reproducibility metrics of the system are presented, along with comparative analysis of protein targets in mitotic versus asynchronous cells. We forward this basic 2D approach to facilitate wider implementation of top-down mass spectrometry and a variety of other protein separation and/or characterization approaches.

Figure 1

Workflow for top-down proteomics. Total protein content from HeLa-S3 nuclei or cytosol is quantified and loaded onto a gel-elution (GE) column. The GE device separates the protein samples according to molecular weight (MW). Proteins of increasing MW elute into solution-phase fractions, which can be visualized on a slab gel (top right). The solution-phase fractions are cleaned up to remove SDS before injection onto a nano-liquid chromatography (LC) column for tandem mass spectrometric (MS/MS) analysis with an LTQ-FT at either 12 or 14.5 T. LC-MS/MS files are processed with ProSightPC 2.0, a software suite tailored for top-down analysis in a high-throughput setting.

Figure 2

Examples selected from an LC-MS/MS injection of fraction #3 from a Tris-glycine GE run. (a) A base-peak chromatogram is shown, with (b) charge state distributions from selected retention times. (c) Abundant charge states (above the arrows) were targeted for fragmentation. Fragmentation mass spectra for each protein are shown along with the corresponding identifications and E-values. A fragmentation map (d) results from the matching fragment ions of nucleoside diphosphate kinase B. The protein is N-terminally acetylated.

Figure 3

Analytical slab gel and heat map showing masses detected in GE fractions. The silver-stained slab gel (top) of solution-phase fractions shows typical GE separation (Tris-tricine buffer system). After LC-MS of the GE fractions, the detected masses for each fraction are plotted as a function of LC retention time (bottom). The MS relative abundance is designated with the scale on the right; the inset illustrates the enlarged region of heat map of GE fraction 6. General correlation can be seen between detected masses and abundances from the MS and the intensities of protein bands detected on the slab gel.

Figure 4

Differences in phosphorylation for proteins identified in asynchronous and M-phase arrested HeLa cells. (a) Asynchronous HeLa proteins are identified with the phosphorylations designated in red. (b) M-phase arrested HeLa proteins are identified, again with phosphorylations designated in red. Dynamic changes in phosphorylation between asynchronous and M-phase arrested HeLa cells were clearly observed. (c) A fragment map for the doubly phosphorylated 60S acidic ribosomal protein (phosphorylation sites in red text).