Allergen Shedding in Human Milk from Mothers of Preterm Infants: Proteomic and Peptidomic Feasibility and Pilot Analysis

Rationale: There is little information regarding the allergen content of milk feeds in the preterm population. Previous studies have evaluated specific proteins/peptides via ELISA, but no studies have performed a broad analysis of the allergenic peptide content and protease activity of milk feeds in this population. Preterm infants spend a critical window of time for immune development in the Newborn Intensive Care Unit (NICU), and may receive fortified donor milk, maternal milk or formula feeds via nasogastric tube or bottle instead of fresh breastmilk via breastfeeding. Methods: To evaluate feasibility, we initially performed mass spectrometry on four human milk samples (two term and two preterm) from the Mommy’s Milk Human Milk Biorepository (HMB) which included maternal surveys of diet and environmental exposures. We analyzed the results against the University of Nebraska FASTA database and UniProt for a total of 2211 protein sequences. We then further analyzed 5 samples from the Microbiome, Atopy and Prematurity (MAP) pilot study along with formula and human milk fortifier controls and performed not only mass spectrometry, but also peptidomic and protease activity analysis. Results: Each HMB sample had between 806 and 1007 proteins, with 37 to 44 non-human proteins/sample encompassing 26 plant and animal species. Bovine proteins were the most numerous; seven unique Bos taurus proteins were found in all four samples, and three contained Bos d 5. Cat, dog, mosquito, salmon, and crab were detected in all four samples. All donors ingested fish, shellfish and tree nuts, and all had salmon and crab proteins in their milk samples; two almond proteins were detected in three samples. Aeroallergens, including dust mite (Der f 28, Der f 25) and mold (Cla h 4) were identified in all samples. Two samples contained allergens to latex (Hev b 9) and chicken (Gal d 10). One sample contained several unique proteins, including carrot, two molds (including Pen c 19) and Der f 33-like protein. In the preterm MAP samples, 784 digested non-human proteins were identified, 30 were non-bovine in origin. Proteins from 23 different species including aeroallergens, food, and contact allergens were identified. Protease activity was highest in human milk samples without human milk fortifier and lowest in preterm formula. Conclusions: These findings represent the first preterm milk feed mass spectrometry and protease analysis with identification of known allergenic proteins to food, contact and aeroallergens. The varying degree of protein detection may reflect variable individual secretion and augmentation of feeds. This raises questions of whether the composition of milk feeds in the NICU impact the development of atopic disease in the preterm population and whether the complex interaction between allergens, proteases, and other human milk components can serve to induce sensitization or tolerance to allergens in infants.