GLOBAL PROTEOMIC ANALYSIS OF TRANSPORTER PROTEINS IN THE HUMAN SMALL INTESTINE AND ESTABLISHED INTESTINAL CELL LINES

The efficacy and safety of many drugs is markedly affected by the expression and function of intestinal uptake and/or efflux transporters. In contrast to the liver, which is well characterized in terms of expression and function of drug transporters and their interplay with metabolizing enzymes, the respective knowledge is much more limited for the human intestine although most drugs on the market are administered via oral route. Moreover, available studies were almost exclusively focused on a very limited number of proteins such as ABCB1 (P-gp) and ABCC2 (MRP2).

Thus, it was the aim of this study to characterize the global transporter profile in human intestinal tissue and intestinal cell lines that are frequently used in preclinical drug development. In addition, these results will be compared with data from targeted proteomics assays for clinically relevant transporters.

For this purpose, tissue samples from jejunum (N=5, 2 males, 3 females, age: 48-81) and ileum (N=5, 4 males, 1 female, age 58-80) and cell pellets from LS180, FHs74Int and Caco-2 cells were collected and the plasma membrane fraction was isolated by standard methods. Afterwards, the samples were subjected to a tryptic digest (1:100, 16 h) before undergoing mass spectrometry (MS)-based analyses. A shotgun proteomics approach was applied for assessing the transporter expression pattern using a 2 h chromatographic gradient on a nano HPLC system coupled to a QExactive plus MS. The MS data were analysed with the MaxQuant 1.5.8.0 software. Targeted proteomics analysis was done on a Sciex QTRAP 5500 using validated methods.

In human intestinal tissue and the investigated intestinal cell lines about 3000-4000 proteins could be identified. With regard to transport proteins, the global analysis could identify 18 ABC and 116 SLC transporters in the jejunum and 17 ABC and 103 SLC in the ileal samples. In addition to well-known intestinal proteins such as ABCB1, ABCC2/3, ABCG2 and PEPT1 several additional and so far not reported proteins could be identified the abundance of which was with the exception of few examples (e.g. ABCB1, OSTalpha) in most cases higher in jejunum than in ileum. In the intestinal cell lines, considerably fewer transport proteins could be identified than in the small intestine (Caco-2, 14 ABCs, 79 SLCs; LS180, 7 ABCs, 47 SLCs; FHs74Int, 14 ABCs and 80 SLCs). The protein amounts of detected transporter proteins varied substantially between the different cell lines and were not superimposable with data that have been observed in the human intestinal segments.

In conclusion, our study characterized for the first time the global transporter protein profile in the human small intestine and in frequently used intestinal cell lines. These data may be useful for identifying so far not considered proteins. In addition, the combination of these results and absolute quantitative data may contribute to a better understanding of intestinal drug absorption. Furthermore, the transporter expression patterns that have been observed in the intestinal cell lines differed substantially from those of human tissue samples which should be considered in in vitro in vivo correlations.