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Letters to the Editor |
1 Department of Medicine, The University of Sydney, New South Wales, Australia
2 INSERM U836, Department of Proteomics, Grenoble, France
3 Department of Pediatrics, KCHRI, and 4
Department of Pharmacology and Toxicology University of Louisville, KY
5 INSERM ERI17 Grenoble, France
6 Faculté de Médecine, Université Joseph Fourier, Grenoble, France
7 CHU, Hôpital A. Michallon, Laboratoires du sommeil et EFCR, Grenoble, France
aAddress correspondence to this author at: Laboratoire HP2, Université Grenoble 1, Institut Jean Rojet, BP170, 38042 Grenoble Cedex 09, France. Fax 33 4 76637178; e-mail maurice.dematteis{at}ujf-grenoble.fr.
To the Editor:
Bouamrani et al. (1) recently reported an approach to SELDI-TOF mass spectrometry that uses direct apposition of cryosections onto proteinchips to enrich spectral profiles and improve the discriminatory power of the technique. To prevent protein degradation, however, samples must be stored frozen and locally processed or shipped frozen to a proteomics facility. We have found that tissue can be preserved on filter paper in a way that maintains the protein profiles obtained with frozen tissues and allows targeted tissue analysis and storage at room temperature.
We use a type of filter paper routinely used for body fluid sample collection, transport, and archiving for various analyses (2). We used serial coronal sections from frozen rat brains at the caudate-putamen (CP) level (anteroposterior: 0.70 mm from bregma) to compare nonpaper- and paper-collection methods. Nonpaper methods comprised 2 different techniques, tissue apposition (TA) and tissue lysate (TL). For TA, 4 mm2 of the CP area was isolated from 10-µm cryosections and apposed onto an NP20 proteinchip. For TL, a similar 4-mm2 sample was inserted into an Eppendorf tube and 20 µL lysis buffer (LB) or distilled water (dH2O) was added, vortex-mixed (30 s), left 10 min on ice, centrifuged, and then 2 µL of the supernatant was pipetted onto the protein chip.
The paper methods also compared the 2 techniques of apposition and lysate. Cryosections (20 µm) were collected onto a filter paper (903-paper®, Whatman) and dried 2 h at room temperature. Then we used a micropuncher to remove 2-mm micropunches from the CP. For paper apposition (PA), the micropunch was applied onto adhesive tape and briefly rehydrated (60 s) with 1 µL LB or dH20 on its tissue-side before apposing onto the proteinchip. For paper lysate (PL), the micropunch was processed in a tube similarly to TL, and 2 µL of the supernatant was pipetted onto the proteinchip. We compared LB and dH2O protein extraction protocols because protein profiles may depend on the elution buffer (2). We assessed protein stability on paper by collecting and drying tissue as above and packing the paper in a storage bag with desiccant sachets before storing it for 30 days in a drawer at room temperature. Sinapinic (SPA) matrix was applied to the proteinchip arrays before reading with the Ciphergen ProteinChip® Reader PCS4000 (6000 nJ laser energy). Data were analyzed with Ciphergen Express software. Repeatability and reproducibility were assessed with 2 sets of duplicate samples run on consecutive days, calculating intra- and interassay CVs. A detailed protocol is available on request.
No major differences were found between TA and PA or TL and PL, suggesting that the 100% pure cotton linters paper did not interfere with the analysis. The closest mass spectral pattern to TA was obtained with PA (Fig. 1
, A and B). In contrast, TL and PL showed fewer peaks within the 4–8 kDa range (Fig. 1A
) in agreement with previous reports (1)(2), and lower peak intensities with most peaks barely above background noise resulting in inaccurate peak labeling (Fig. 1
, A and B). In addition to spectral enrichment, PA was the quickest method (1–2 min vs 45 min for PL), allowing easy handling of thin tissue sections and micropunching to collect replicate selections of minute regions of interest. This method simplifies targeted tissue analysis for tissue proteomic mapping compared to molecular histology(3) or proteohistography(4). The PA method also demonstrated repeatable and reproducible measurements with an intra- and interassay pooled CV <0.06% for m/z, and between 6.9% and 14% for intensity (Fig. 1C
), these values are well within the acceptable range(5). Dried paper stored at room temperature for 30 days gave similar spectra to those obtained from freshly processed papers (Fig. 1D
). The absence of protein denaturation in air-dried samples without protease inhibitors may stem from the absorption/drying characteristics of the 903-paper. Regarding protein recovery, the paper rehydration with dH2O led to spectra similar to those of LB (Fig. 1E
, upper panel). In contrast, dH2O protein extraction for the TL method resulted in spectra with more peaks of greater amplitude than with LB (Fig. 1E
, lower panel). The fact that dH2O is more acidic than LB may have altered the ionization process and the resulting spectrum(2). In contrast, the rapid paper rehydration with only 1 µL solution may have minimized chemical interactions.
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In summary, this simple, time-efficient method for collection and targeted analysis allows room temperature storage of tissue for at least 30 days. This method may facilitate shipping and sharing between laboratories, and it opens new perspectives in clinical and experimental proteomics, as well as in genomics and transcriptomics.
Acknowledgments
Grant funding/support: This research was supported by a grant from AGIR@dom and NIH P20 RR-15576. Dr. Machaalani was supported by a travel grant through Human Frontier Science Program (HSFP) to conduct this research in France.
Financial disclosures: None declared.
References
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