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. 2021 Jun 14;11:12427. doi: 10.1038/s41598-021-91684-4

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© The Author(s) 2021

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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(A) Flow diagram showing collection and processing of samples from three different physiological states of Sahiwal cow followed by separation of small-sized peptides (<10 kDa) by ultrafiltration assembly and its extraction by C18 solid-phase extraction method. (B) The solid phase extraction (SPE) method. The bound analytes in the column were first eluted in 60% ACN, 0.1% TFA followed by the extraction of peptides in the aqueous phase using ethyl acetate. An equal amount of peptides from each sample (biological replicates) was pooled group-wise and quantified, for nLC-MS/MS analysis in triplicate (technical replicates).