Lauren M. Dimapasoc; Sarah Totten; Carol Stroble; L. Renee Ruhaak; Carlito B. Lebrilla

University of California, Davis, CA
NOVEL ASPECT: HMO extraction and purification at the 96-well level now allows for large-scale biomarker studies.
Human milk is known to be the best nutritional source for newborns. Human milk oligosaccharides (HMOs) are a highly abundant constituent in human milk, and its protective and prebiotic effects are what drive studies today. Further investigating these properties and future biomarker studies will require large patient sample sets, where high-throughput sample preparation and analysis are favored. The development of a sample preparation method at the 96-well level would increase the repeatability of the process and reduce batch-to-batch sample variations. Furthermore, it will allow shorter handling times, giving us the ability to process larger amounts of samples. In this study, we have developed and optimized a high-throughput method to rapidly profile human milk oligosaccharides at a 96-well level.
Five batches of breast milk samples from the same donor were processed to find the optimal separation method by using the traditional HMO extraction method under different conditions (exclusion of ethanol, Folch, and precipitation times). Samples were reduced and purified using an automated liquid handler to prevent variation in solid phase extraction (SPE). Then the optimized method was repeated using smaller volumes, and SPE on a 96-well plate with graphitic carbon was administered via centrifugation to test the saturation limit of the columns. Glycan profiles and isomer information were obtained by HMO analysis on a chip-based nano-LC (Agilent Chip/TOF-MS) using acetonitrile/water and formic acid for separation. For data-processing, an in-house library of HMO masses was used.
Based on the HMO extraction and purification method that was previously established in our lab, we first optimized whether we could omit certain steps that may be difficult to process at the 96-well plate level. Traditionally, human milk is centrifuged to remove fats, treated with chloroform/methanol (Folch) to remove additional lipids, and treated with ethanol to remove excess proteins prior to reduction and SPE. After we excluded the Folch method, the overall HMO profile was still comparable to samples treated with Folch using 500uL of sample. However, excluding both the Folch and ethanol addition resulted in lower analytical signal and different HMO profiles compared to the traditional batch.We then evaluated a commercially available 96-well graphitic carbon SPE plate and compared it to graphitized carbon cartridges used for individual sample analysis to test the saturation limit of the 96-well plate. To accommodate the 96-well plate, the sample volumes were reduced to 10, 25, 35, and 50uL and SPE was performed using centrifugation. While solvent amounts were reduced for plate-based SPE, no additional contaminants were observed during the analyses. Samples were reconstituted to 50uL to show a gradual increase in glycan signal between the different volumes and compositional glycan profiles were obtained by Agilent Chip/TOF-MS and identified using an in-house library of accurate glycan masses found in humans. It could be concluded that the 96-well graphitic carbon plate is not saturated using the milk volumes used.With this procedure, we are able to process 96 samples with a handling time of around 3 hours. The application of 96-wells allows for reduction of batch-to-batch sample variations, and increases repeatability of the sample preparation procedure. This procedure will allow the processing of large sample cohorts to further investigate the biological activity of HMOs and how it may possibly be changing in case studies.