Comprehensive identification of proteins from MALDI imaging

Abstract

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is a powerful tool for the visualization of proteins in tissues and has demonstrated considerable diagnostic and prognostic value. One main challenge is that the molecular identity of such potential biomarkers mostly remains unknown. We introduce a generic method that removes this issue by systematically identifying the proteins embedded in the MALDI matrix using a combination of bottom-up and top-down proteomics. The analyses of ten human tissues lead to the identification of 1400 abundant and soluble proteins constituting the set of proteins detectable by MALDI IMS including >90% of all IMS biomarkers reported in the literature. Top-down analysis of the matrix proteome identified 124 mostly N- and C-terminally fragmented proteins indicating considerable protein processing activity in tissues. All protein identification data from this study as well as the IMS literature has been deposited into MaTisse, a new publically available database, which we anticipate will become a valuable resource for the IMS community.

Fig. 1.

Extraction of proteins from MALDI IMS slides. A, the sinapinic acid matrix layer of a ready-to-measure MALDI IMS microscopy slide (here prepared from normal stomach tissue is removed by a two-step extraction procedure using 7.5 and 60% acetonitrile in 0.2% tri-fluoro acetic acid (TFA). The remaining tissue is recovered by means of a scalpel (see also supplemental Fig. S1A) B, silver stained SDS-PAGE of the proteins recovered from the microscopy slide showing that the acetonitrile extractions primarily recover low molecular weight proteins (see also supplemental Fig. S1B).

Fig. 2.

Bottom-up proteomic characterization of MALDI IMS samples. A, protein identification summary of ten human tissues. B, cumulated number of identified proteins from the ten different tissues and three protein extractions. Samples are ordered such that the highest number of identifications is obtained for the smallest number of samples combined. C, distribution of identified proteins by molecular weight. Bins were chosen according to the marker used in the SDS-PAGE shown in Fig. 1B. Blue bars represent the distribution of entries in the Uniprot database (human only). D, distribution of proteins by cellular compartment.

Fig. 3.

Relative abundance of proteins identified in MALDI IMS. A, Proteins from all 10 human tissues were ranked according to the number of assigned spectra divided by the molecular weight of the proteins' database entry. MALDI IMS markers extracted from the literature (see also Table I and supplemental Table S5) are indicated in orange showing that these are mainly abundant cellular proteins. B, Proteins identified from the MALDI matrix layer plotted against the ranked protein abundance used in panel A).

Fig. 4.

Top-down protein identification. A, charge deconvoluted and deisotoped tandem mass spectrum of intact and acetylated thymosin beta-4. (only b and y ions are shown, for a completely annotated spectrum see supplemental Fig. S3). B, comparison of top-down protein identifications made by searching the human IPI database (87,061 entires) or subsetDB (5455 entires) showing that almost all proteins can be identified in the 15x smaller subsetDB. C, distribution of precursor ion masses leading to a successful top-down identification. D, normalized sequence coverage plot of top-down identified proteins in which each line represents one protein. The darker the gray scale density, the more spectra matched to the respective region of the protein indicating that most proteins identified from MALDI IMS samples are fragments of the original protein sequence.