T(3): targeted proteomics of DNA-binding proteins

Abstract

A technique that allows the inclusion of a specific DNA to enrich and direct proteomic identification of transcription factors (TFs) while providing a route for high-throughput screening on a single platform would be valuable in investigations of gene expression and regulation. Polyvinylpyrrolidone binds DNA avidly while binding negligible amounts of protein. This observation is used in a proof-of-concept method to enrich for TFs by combining nuclear extract with a specific DNA sequence and immobilizing the DNA-protein complex on a polyvinylpyrrolidone (PVP)-coated MALDI (matrix-assisted laser desorption/ionization) plate. Any unbound proteins are washed away and further processed for analysis in a MALDI-TOF/TOF (tandem time-of-flight) mass spectrometer. Enrichment on a PVP-coated plate gives the unique advantage of purification, enzymatic digestion, and analysis on a single platform. The method is termed T(3) because it combines Targeted purification on a Target plate with Targeted proteomics. Validation was achieved in model experiments with a chimeric fusion protein, green fluorescent protein-CAAT enhancer binding protein (GFP-C/EBP), with an oligonucleotide containing the CAAT sequence. Both domains were identified with an expectation value of less than 10(-15) and more than 15% sequence coverage. The same oligonucleotide mixed with HEK293 cell nuclear extract allowed the unambiguous identification of native human C/EBP alpha with 24.3% sequence coverage.

Keywords: DNA-binding proteins; MALDI–MS; Mass spectrometry; Proteomics; Transcription factors.

Figure 1. T³ Schematic

Adsorption of PVP to the MALDI plate was accomplished by immersing the plate in 25 mg PVP/100 mL methanol and incubated undisturbed for 48 hours under N₂. An oligonucleotide containing a sequence of interest was mixed with protein under conditions that encourage DNA-protein complex formation. Then the solution was spotted onto the PVP-coated-target-plate. After incubation, each spot was washed twice with binding buffer to remove any non-specific proteins and then washed once with diH₂O to remove salts present in the binding buffer. Reduction, alkylation and trypsin digestion was carried out on the target plate, then matrix was applied to the digested sample and analysis was accomplished by MALDI-TOF-MS/MS.

Figure 2. Uncoated Plate vs. Coated Plate, with and without DNA

The T³ technique was evaluated on an uncoated plate (panel A), or a coated plate (panels B and C). In panel B, 10 nM DNA was mixed with the protein, while in panel C DNA was omitted. All panels have a peak at m/z = 842.5 which corresponds to trypsin sequence VATVSLPR. In panel B, 13 peptides were identified by MALDI-TOF-MS/MS to arise from GFP-C/EBP. Note: each panel in Fig. 2 is not shown at the same intensity; with the lowest being panel A.

Figure 3. Annotated Spectrum of Green Fluorescent Protein

The GFP region (239 amino acid, N-terminal) received 31% sequence coverage and an ion score of 497.72 by Mascot search engine and SwissProt database. In the upper panel, the peaks with masses labeled corresponds fragments from tryptic peptide DDGNYKTR, matching GFP after CID of m/z = 1604.81. In the lower panel, the numbers to the right of the sequences represents the residue number. Grey lettering denotes plasmid-derived sequences, green represents sequences derived from GFP, and black lettering are from C/EBP sequences. Bold and italicized lettering specifies the different sequences determined by MALDI-MS/MS.

Figure 4. Annotated Spectrum of CAAT Enhancer Binding Protein

The C/EBP region (101 amino acid, C-terminal) was identified with 23.8% sequence coverage and ion score of 159.86 by protein database searching with Mascot software and SwissProt database. The upper panel represents a spectra obtained from CID of m/z = 1622.83, the peaks with labeled masses denotes fragments from tryptic peptide matching RRVRYENSNK. In the lower panel, the coloring scheme is the same as Fig. 3. Identifications were all found on the C-terminus C/EBP sequences of this chimera.

Figure 5. EP18 and BSA used in Oligo-MALDI-MS

A PVP coated plate with 10 nM EP18 oligonucleotide, 2.8 mg/ml bovine serum albumin, and CHCA matrix with an increased laser intensity of 80%.

Figure 6. Mass Spectrum of EP18 oligonucleotide on PVP coated MALDI plate

Detection of the oligonucleotide allows confirmation of the adsorbed DNA. DNA sequence (5’-GCAGATTGCGCAATCTGA-3’) was detected with a +1 charge and m/z = 5483.8 using 3-hydroxypicolinic acid as the matrix. The calculated mass for this DNA sequence is 5482.8.

Figure 7. Spectrum of Green Fluorescent Protein and CCAAT Enhancer Binding Protein purified from spiked nuclear extract

The 5 µl sample spotted on the plate contained 10 nM EP18 oligonucleotide and 5 µg HEK293 nuclear extract and 0.35 µg of purified GFP-C/EBP. The GFP region (239 amino acid, N-terminal) was identified with a Mascot protein score of 166 and 15.5% sequence coverage. The C/EBP region (101 amino acid, C-terminal) was identified with 17.8% sequence coverage and a Mascot protein score of 63. Protein database were searched with Mascot software and SwissProt database. In the upper panel, the peaks with green-labeled masses denote peptides derived from GFP and black-labeled masses correspond to C/EBP by MALDI-MS. In the lower panel, the coloring scheme is the same as Fig. 3.

Figure 8. Sequence Coverage of Native CAAT Enhancer Binding Protein Enriched from Nuclear Extract

Human C/EBP alpha was identified with 24.3% sequence coverage and a Mascot protein score of 38 by protein database searching with Mascot software and SwissProt database. Peaks with masses labeled correspond to tryptic peptides matching C/EBP. High sequence coverage and low protein score can be explained by the targeted approach and the spectra collected having low signal to noise.

Figure 9. Comparison of MS/MS Spectra of CAAT Enhancing Binding Protein Enriched from Spiked and Un-Spiked Nuclear Extract

5 µl samples spotted contained 10 nM EP18 oligonucleotide and either (A) 5 µg NE spiked with 0.35 µg GFP-C/EBP or (B) un-spiked NE. Spectra were collected in positive LIFT mode using CHCA matrix. MS/MS of m/z 1127.6 corresponds to sequence RERNNIAVR. The position of fragments in the overall sequence is shown in the scheme at the top of the spectrum.