Proteomics 2016, 16, 1975–1979
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DOI 10.1002/pmic.201500385
TECHNICAL BRIEF
High performance CCD camera system for digitalisation of 2D DIGE gels Annemieke Strijkstra1,2∗ , Kathleen Trautwein1,2∗ , Stefan Roesler3 , Christoph Feenders4 , Daniel Danzer3 , Udo Riemenschneider3 , Bernd Blasius4 and Ralf Rabus1,2 1
General and Molecular Microbiology, Institute for Chemistry and Biology of the Marine Environment (ICBM), Carl von Ossietzky University Oldenburg, Oldenburg, Germany 2 Department of Microbiology, Max Planck Institute for Marine Microbiology, Bremen, Germany 3 ¨ Intas Science Imaging Instruments GmbH, Gottingen, Germany 4 Mathematical Modelling, Institute for Chemistry and Biology of the Marine Environment (ICBM), Carl von Ossietzky University Oldenburg, Oldenburg, Germany
An essential step in 2D DIGE-based analysis of differential proteome profiles is the accurate and sensitive digitalisation of 2D DIGE gels. The performance progress of commercially available charge-coupled device (CCD) camera-based systems combined with light emitting diodes (LED) opens up a new possibility for this type of digitalisation. Here, we assessed the performance of a CCD camera system (Intas Advanced 2D Imager) as alternative to a traditionally employed, highend laser scanner system (Typhoon 9400) for digitalisation of differential protein profiles from three different environmental bacteria. Overall, the performance of the CCD camera system was comparable to the laser scanner, as evident from very similar protein abundance changes (irrespective of spot position and volume), as well as from linear range and limit of detection.
Received: September 24, 2015 Revised: April 20, 2016 Accepted: May 31, 2016
Keywords: 2D DIGE / CCD camera / Laser scanner / Intas Advanced 2D Imager / Technology / Typhoon
Additional supporting information may be found in the online version of this article at the publisher’s web-site
Proteomic research within the past two decades has revealed 2D DIGE as gold standard for gel-based, high confidence analysis of protein abundance changes (e.g. [1, 2]). Key requirement for capturing the broad dynamic range and context-specific changes in protein abundances is the precise and sensitive digitalisation of 2D DIGE gels. This has been achieved by laser scanners employing confocal optics (Fig. 1A), with the TyphoonTM three-mode laser scanner (GE Healthcare, Munich, Germany) representing the benchmark instrument for 2D DIGE gel image digitalisation during the Correspondence: Professor Ralf Rabus, General and Molecular Microbiology, Institute for Chemistry and Biology of the Marine Environment (ICBM), Carl von Ossietzky University Oldenburg, Carl-von-Ossietzky Str. 9–11, D-26111 Oldenburg, Germany E-mail:
[email protected] Fax: +49-441-798-3404 Abbreviations: CCD, charge-coupled device; LED, light emitting diode
past 15 years. Gel-based differential proteomics could benefit largely from commercially available and sensitive, 2D DIGE-suited CCD camera systems (Fig. 1B) that would allow for strongly reduced image acquisition times as well as instrument maintenance costs, resulting from the simple and robust design. In this study, we tested the prototype of a CCD camera system (Intas Advanced 2D Imager; Intas Science Imaging Instruments GmbH, G¨ottingen, Germany) (Fig. 1B) and compared its performance to the Typhoon 9400 laser scanner. Experiments involved the proteomically well-characterised, environmental bacteria “Aromatoleum aromaticum” EbN1 [3], Desulfobacula toluolica Tol2 [4], and Phaeobacter inhibens DSM 17395 [5]. For each bacterial strain, four replicate 2D DIGE ∗ These authors contributed equally. Colour Online: See the article online to view Figs. 1–3 in colour.
[The copyright line of this article has been changed since first published on 2 June 2016 from the standard copyright to CC-BY-NC-ND.]
C 2016 The Authors. Proteomics Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
www.proteomics-journal.com This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
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Proteomics 2016, 16, 1975–1979
Figure 1. Schematic representation of the compared digitalisation systems. (A) Typhoon laser scanner. For excitation, CyDye-specific laser beams are individually delivered from the respective laser source via optical fibre cables through the scan head (positioned under the gels), and optically focused into the gel plane. Fluorescent light emitted from the gel passes through dye-specific bandpass filters and reaches the detector via another optical fibre cable. The described excitation/emission process occurs repeatedly at high spatial resolution (typically 100 m). (B) CCD camera system. Two oppositely positioned LED arrays with identical dye-specific filters, forming one LED module, directly and evenly illuminate the complete area of the 2D DIGE gel (21 × 27 cm size) at an optimised angle (Supporting Information Fig. S1). The emitted light from the entire gel passes through a dye-specific emission filter in front of the camera optics. A 16-bit CCD captures, amplifies and converts the light signal via an electric current into 65 536 digital grey scale values.
gels of 21 × 27 cm size were digitalised with both imaging systems to comparatively evaluate instrument performances based on protein spot detection, spot volumes and abundance changes (average ratios). In addition, 1DE gels were used to determine the spectral cross-contamination in the emission of the employed fluorophores (CyDyesTM ; GE Healthcare), as well as the limit and linear range of detection. Cultivation of bacterial strains was performed as previously described [4–6]. Preparation of soluble proteins, determination of protein concentrations and all experimental procedures of 2D DIGE (minimal labelling with 200 pmol CyDye per 50 g protein), digitalisation with the laser scanner and image analysis with the DeCyderTM 2D software (version 7.0; GE Healthcare) were performed as previously described in detail [1]. Protein extracts from cells grown with 4-ethylphenol (strain EbN1), toluene (strain Tol2), or casamino acids (strain DSM 17395) represent three different test states and were each labelled with Cy3. Protein extracts from each bacterial strain grown with fumarate served as reference state and were independently labelled with Cy5. Three strain-specific internal standards were prepared from equal amounts of the corresponding test and reference state and were each labelled with Cy2. An individual gel contained 50 g each of labelled reference and test state, and of the internal stan-
dard. For each strain, four parallel gels were prepared with protein samples from four individual cultures to account for biological variation [7]. Isoelectric focusing (IEF) was conducted using 24 cm-long IPG strips (GE Healthcare) with a nonlinear pH range from 3 to 11 (for strains EbN1 and Tol2) or 3–5.6 (for strain DSM 17395) in a Protean i12 system (Bio-Rad, Munich, Germany). The IEF program used was as follows: 50 V for 13 h, 200 V for 1 h, 1000 V for 1 h, gradual gradient to 10 000 V within 2 h, and 10 000 V for 6 h. Separation of proteins according to molecular mass was done by SDS-PAGE (12.5% acrylamide, v/v) using an EttanDalttwelve (GE Healthcare) system. 2D DIGE gels were digitalised directly after electrophoresis remaining sandwiched between two thoroughly cleaned and dry low-fluorescence glass plates (GE Healthcare). First, 2D DIGE gels were digitalised with the CCD camera system and then immediately with the laser scanner; gels in standby position were stored at 4⬚C in the dark. In case of the CCD camera system, individual gels were precisely positioned on a black glass tray (CAS 65997-17-3), residing on a size-matched, elevated aluminium platform (Fig. 1B). All interior surfaces of the steel cabinet are powdercoated with a non-fluorescent, highly light-absorbing material. The optical field and focus plane were manually adjusted
C 2016 The Authors. Proteomics Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
www.proteomics-journal.com
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Proteomics 2016, 16, 1975–1979 Table 1. Results of the DeCyder 2D-based image analysis
Individual workspaces in DeCyder 2D analysis “A. aromaticum” EbN1
D. toluolica Tol2
P. inhibens DSM 17395
Laser scanner CCD camera Laser scanner CCD camera Laser scanner CCD camera No. of detected spotsa) By automatic detection After manual refinement No. of matched spots No. of congruent spots analysed per bacterial strain (793 in total)b) Average ratio 1.5 Average ratio
