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样品制备方法对高效液相色谱-串联质谱分析人乳内源肽的影响

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 spe...

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Autores principales: YU, Wenhao, YU, Yang, WANG, Wendan, LI, Yitong, SZETO, Ignatius M., JIN, Yan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Editorial board of Chinese Journal of Chromatography 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9403996/
https://www.ncbi.nlm.nih.gov/pubmed/34227330
http://dx.doi.org/10.3724/SP.J.1123.2020.08019
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author YU, Wenhao
YU, Yang
WANG, Wendan
LI, Yitong
SZETO, Ignatius M.
JIN, Yan
author_facet YU, Wenhao
YU, Yang
WANG, Wendan
LI, Yitong
SZETO, Ignatius M.
JIN, Yan
author_sort YU, Wenhao
collection PubMed
description 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 β-casein, polymeric immunoglobulin receptor, osteopontia, α(S1)-casein, κ-casein, and bile salt-activated lipase were identified in all samples extracted using the six methods. Moreover, these precursor proteins contributed 88% peptides in the samples extracted using the six methods. In conclusion, UF 1 and UF 2 were efficient procedures for extracting endogenous peptides from human milk. In addition, UF 2 could extract peptides from lactotransferrin, which is the optimum choice for extracting endogenous peptides from human milk. Additionally, the OMC enrichment method can be used to enrich and extract specific endogenous peptides from human milk. This study systematically compared the sample preparation methods commonly used in human milk endogenous peptidomics in recent years. The results provide strong support for uniform and standardized sample preparation methods. An ultrafiltration method without denaturation, which is more advantageous than the currently commonly used trichloroacetic acid precipitation method, was also established to prepare human milk endogenous peptide samples. In combination with OMC, this method can help in a more comprehensive and in-depth understanding of the endogenous peptidome of human milk.
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spelling pubmed-94039962022-09-14 样品制备方法对高效液相色谱-串联质谱分析人乳内源肽的影响 YU, Wenhao YU, Yang WANG, Wendan LI, Yitong SZETO, Ignatius M. JIN, Yan Se Pu Articles 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 β-casein, polymeric immunoglobulin receptor, osteopontia, α(S1)-casein, κ-casein, and bile salt-activated lipase were identified in all samples extracted using the six methods. Moreover, these precursor proteins contributed 88% peptides in the samples extracted using the six methods. In conclusion, UF 1 and UF 2 were efficient procedures for extracting endogenous peptides from human milk. In addition, UF 2 could extract peptides from lactotransferrin, which is the optimum choice for extracting endogenous peptides from human milk. Additionally, the OMC enrichment method can be used to enrich and extract specific endogenous peptides from human milk. This study systematically compared the sample preparation methods commonly used in human milk endogenous peptidomics in recent years. The results provide strong support for uniform and standardized sample preparation methods. An ultrafiltration method without denaturation, which is more advantageous than the currently commonly used trichloroacetic acid precipitation method, was also established to prepare human milk endogenous peptide samples. In combination with OMC, this method can help in a more comprehensive and in-depth understanding of the endogenous peptidome of human milk. Editorial board of Chinese Journal of Chromatography 2021-05-08 /pmc/articles/PMC9403996/ /pubmed/34227330 http://dx.doi.org/10.3724/SP.J.1123.2020.08019 Text en https://creativecommons.org/licenses/by/4.0/本文是开放获取文章,遵循CC BY 4.0协议 https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Articles
YU, Wenhao
YU, Yang
WANG, Wendan
LI, Yitong
SZETO, Ignatius M.
JIN, Yan
样品制备方法对高效液相色谱-串联质谱分析人乳内源肽的影响
title 样品制备方法对高效液相色谱-串联质谱分析人乳内源肽的影响
title_full 样品制备方法对高效液相色谱-串联质谱分析人乳内源肽的影响
title_fullStr 样品制备方法对高效液相色谱-串联质谱分析人乳内源肽的影响
title_full_unstemmed 样品制备方法对高效液相色谱-串联质谱分析人乳内源肽的影响
title_short 样品制备方法对高效液相色谱-串联质谱分析人乳内源肽的影响
title_sort 样品制备方法对高效液相色谱-串联质谱分析人乳内源肽的影响
topic Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9403996/
https://www.ncbi.nlm.nih.gov/pubmed/34227330
http://dx.doi.org/10.3724/SP.J.1123.2020.08019
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