MALDI MS Imaging of Chickpea Seeds (Cicer arietinum) and Crab's Eye Vine (Abrus precatorius) after Tryptic Digestion Allows Spatially Resolved Identification of Plant Proteins.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) imaging following in situ enzymatic digestion is a versatile analytical method for the untargeted investigation of protein distributions, which has rarely been used for plants so far. The present study describes a workflow for in situ tryptic digestion of plant seed tissue for MALDI MS imaging. Substantial modifications to the sample preparation procedure for mammalian tissues were necessary to cater to the specific properties of plant materials. For the first time, distributions of tryptic peptides were successfully visualized in plant tissue using MS imaging with accurate mass detection. Sixteen proteins were visualized and identified in chickpea seeds showing different distribution patterns, e.g., in the cotyledons, radicle, or testa. All tryptic peptides were detected with a mass resolution higher than 60,000 as well as a mass accuracy better than 1.5 ppm root-mean-square error and were matched to results from complementary liquid chromatography-MS/MS (LC-MS/MS) data. The developed method was also applied to crab's eye vine seeds for targeted MS imaging of the toxic protein abrin, showing the presence of abrin-a in all compartments. Abrin (59 kDa), as well as the majority of proteins visualized in chickpeas, was larger than 50 kDa and would thus not be readily accessible by top-down MS imaging. Since antibodies for plant proteins are often not readily available, in situ digestion MS imaging provides unique information, as it makes the distribution and identification of larger proteins in plant tissues accessible in an untargeted manner. This opens up new possibilities in the field of plant science as well as to assess the nutritional quality and/or safety of crops.

Autofocusing MALDI MS imaging of processed food exemplified by the contaminant acrylamide in German gingerbread.

Acrylamide is a toxic reaction product occurring in dry-heated food such as bakery products. To meet the requirements laid down in recent international legal norms calling for reduction strategies in food prone to acrylamide formation, efficient chromatography-based quantification methods are available. However, for an efficient mitigation of acrylamide levels, not only the quantity, but also the contaminant's distributions are of interest especially in inhomogeneous food consisting of multiple ingredients. A promising tool to investigate the spatial distribution of analytes in food matrices is mass spectrometry imaging (MS imaging). In this study, an autofocusing MALDI MS imaging method was developed for German gingerbread as an example for highly processed and instable food with uneven surfaces. Next to endogenous food constituents, the process contaminant acrylamide was identified and visualized keeping a constant laser focus throughout the measurement. Statistical analyses based on relative acrylamide intensities suggest a higher contamination of nut fragments compared to the dough. In a proof-of-concept experiment, a newly developed in-situ chemical derivatization protocol is described using thiosalicylic acid for highly selective detection of acrylamide. This study presents autofocusing MS imaging as a suitable complementary method for the investigation of analytes' distributions in complex and highly processed food.

MALDI mass spectrometry imaging: From constituents in fresh food to ingredients, contaminants and additives in processed food.

Mass Spectrometry imaging (MS imaging) provides spatial information for a wide range of compound classes in different sample matrices. We used MS imaging to investigate the distribution of components in fresh and processed food, including meat, dairy and bakery products. The MS imaging workflow was optimized to cater to the specific properties and challenges of the individual samples. We successfully detected highly nonpolar and polar constituents such as beta-carotene and anthocyanins, respectively. For the first time, the distributions of a contaminant and a food additive were visualized in processed food. We detected acrylamide in German gingerbread and investigated the penetration of the preservative natamycin into cheese. For this purpose, a new data analysis tool was developed to study the penetration of analytes from uneven surfaces. Our results show that MS imaging has great potential in food analysis to provide relevant information about components' distributions, particularly those underlying official regulations.