[Effect of sample preparation on analysis of human milk endogenous peptides using liquid chromatography-tandem mass spectrometry].

Hundreds of endogenous peptides were released from milk proteins within the human mammary gland and some of them possess a variety of bioactive functions. Thus, it is important to investigate human milk endogenous peptides for infant health. Peptidomics based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been used to investigate human milk endogenous peptides. Extraction of endogenous peptides from human milk is an essential and key procedure for analyzing human milk peptides using LC-MS/MS. This study aimed to compare methods for extracting endogenous peptides from human milk using LC-MS/MS. Ultrafiltration methods including that not involving denaturation (UF 1), that involving heat denaturation (UF 2), and that involving chemical denaturation (UF 3), precipitation methods using trichloroacetic acid (PCPN 1) and alcohol (PCPN 2), and an enrichment method using highly ordered mesoporous carbon (OMC) were used to extract endogenous peptides from human milk. Extracted endogenous peptides were then analyzed using LC-MS/MS. The samples extracted using UF 1 and UF 2 comprised 1161±8 and 1017±91 endogenous peptides, respectively. More than 70% peptide sequences in each sample extracted using UF 1 and UF 2 overlapped. The results revealed that endogenous peptides extracted using UF 1 and UF 2 showed similar characteristics. UF 1 yielded the highest number of peptides, whereas UF 3 extracted the least number of peptides at 366±18. The number of endogenous peptides extracted using PCPN 1 and PCPN 2 were 779±69 and 876±55, respectively. However, their characteristics were quite different, and only about 50% peptide sequences overlapped. The number of peptides extracted using OMC (549±151) was not remarkable compared with that using other methods. However, the isoelectric point (pI) and grand average of hydropathicity (GRAVY) of the peptides extracted using OMC were different from those extracted using other methods. This method presented no selectivity for the endogenous peptides with different pI and GRAVY and may be used to extract unique peptides from human milk. A total of 205 peptides were commonly identified in the samples using each of the six methods. The percent of shared peptides across the six samples ranged from 13% to 23%. The number of unique peptides in the samples extracted using UF 1 and UF 2 (226 and 228, respectively) were the highest among those extracted using the six methods. The results showed that all six methods could be used to extract endogenous peptides from these high-abundance precursor proteins. A total of 21, 38, and 19 peptides were extracted from lactotransferrin using UF 2, UF 3, and OMC, respectively, and the coverage rates of these peptides in lactotransferrin were 14%, 16%, and 19%, respectively. These three methods could extract the endogenous peptides from lactotransferrin in human milk, but PCPN 1 that has been commonly used in previous studies could not. The peptides from