17th International Mass Spectrometry Conference :: Prague, 2006
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|Session:||Instrumentation and Methodologies for Imaging MS|
|Presentation date:||Thu, Aug 31, 2006|
|Presentation time:||14:30 – 16:00|
Erika Regina Amstalden van Hove1, Stefan Luxembourg1, Sander R. Piersma1, Ron M. Heeren1, Iwona Sobczak-Elbourne2, Ate G. J. van der Zee2, Rainer Bischoff2, Ali R. Vaezzadeh3, Catherine G. Zimmermann-Ivol3, Denis F. Hochstrasser31 FOM-AMOLF, Amsterdam, Netherlands
Correspondence address: Erika Regina Amstalden van Hove, FOM-AMOLF, Kruislaan 407, Amsterdam, 1098 SJ Netherlands.
Keywords: Biomarkers; Digests, Tryptic; Imaging; MALDI.
Novel aspect: Combination of direct imaging-MS and molecular scanner approach for peptide identification in tissue section.
Imaging Mass Spectrometry combines spatial localization and identification of biomolecules in cells and tissue. An indirect imaging technique known as Molecular Scanner is used for protein identification. In this approach proteins from tissues or gels are electro-blotted through a trypsin membrane. The digestion products are subsequently trapped on a capture membrane. Alternatively, a protease can be applied directly on the tissue surface prior to directImaging-MS. The objective of this work is to combine both direct and indirect high resolution MALDI MS imaging for protein identification on 1-D gels and tissues with a novel mass microscope.
For direct imaging MS a 10 µm thick tissue section is washed and coated with HCCA matrix using a pneumatic nebulization approach. A 5 nm layer of gold is applied on top of the crystal layer to limit surface charging. The entire surface is scanned in a series of line scans consisting of approximately 1000 laser shots taken while the sample is continuously moving with a fixed speed of 10 µm/s. For each laser shot a total ion image and a spectrum are synchronously acquired. For higher molecular weight (>10kDa) analysis, the tissue was submitted to a various enzymatic digestion strategies (including the molecular scanner) prior to analysis in the MALDI-TRIFT instrument.
In the analysis of intact cervical tissue (both cancerous and healthy tissues) little high MW protein information could be obtained with the direct imaging MS approach. However, after tryptic digestion on the tissue many peptides were found. Most abundant peptides were identified as fragments from hemoglobin alpha and beta and human serum albumin, and many others are still under investigation. The detail provided by the high-resolution images revealed a number of distinct tissue features. The comparison between healthy and cancer tissue indicates that several proteins are present in both tissues, but the intensity ratios differ dramatically. Molecular scanner experiments with SDS-PAGE gels achieved a successful combination of protein identification and spatial localization. With this method it was possible to identify beta-galactosidase, a 116 kDa protein from the imaging MS data. Crucial to the success of this methodology was the careful application of a thin layer of gold prior to analysis. Results on both Meta-SIMS and MALDI imaging MS will show the complementary nature of the two techniques. Moreover, the resolution of 4 µm on a 200 µm diameter field of view per single laser shot allowed a thorough investigation of the sample preparation protocol. In this work a brief description of the various sample preparation protocols for both direct and indirect imaging MS and their merits will be presented.