Supplemental Tables and Figures: Supplemental Table 1: Details of parent proteins from which peptides were derived

Transcription

1 Electronic Supplementary Material (ESI) for Food & Function. This journal is The Royal Society of Chemistry 2017 Supplemental Tables and Figures: Supplemental Table 1: Details of parent proteins from which peptides were derived Paret Protein names Gene name Peptides (n) Mol. Weight, Parent Protein [kda] Sequence length, Parent Protein β-casein CSN polymeric immunoglobulin receptor PIGR α S1 -casein CSN1S osteopontin SPP κ-casein CSN bile salt-activated lipase CEL clusterin CLU lactotransferrin LTF actin, cytoplasmic 1 ACTB butyrophilin subfamily 1 member A1 BTN1A α-lactalbumin LALBA histone H1.2 HIST1H1C thymosin β-4 TMSB protein zeta/delta YWHAZ protein O-linked-mannose beta-1,4- POMGNT N-acetylglucosaminyltransferase protein epsilon YWHAE

2 Supplemental Tabel 2: Fold change of peptide intensity by GA and LS indicated from volcano plots Gestational Age Lactational Stage Sequence Protein Group Fold Change p-value ADGSRASVDSGSSEEQGGSS PIGR ADGSRASVDSGSSEEQGGSSRA PIGR EESITEYKQKV β-casein ESLSSSEESITEYK β-casein ETIESLSSSEESITEY β-casein ETIESLSSSEESITEYK β-casein ETIESLSSSEESITEYKQ β-casein 1.21 < ETIESLSSSEESITEYKQK β-casein ETIESLSSSEESITEYKQKV β-casein LSSSEESITEYK β-casein PAVVLPVPQPEI β-casein PKAKDTVYT β-casein RETIESLSSSEESITEYKQK β-casein SEESITEYK β-casein SLSSSEESITE β-casein SLSSSEESITEYK β-casein SSEESITEYK β-casein SSSEESITEYK β-casein SSSEESITEYKQKV β-casein GAKTTFDVYTESWAQDPSQENK bile salt-activated lipase ADTRDQADGSRASVD PIGR DSGSSEEQGGSSRALVSTLV PIGR VDSGSSEEQGGSSRA PIGR AKDTVYTKGRVM β-casein EESITEYK β-casein 0.97 < ESLSSSEESITEYK β-casein ETIESLSSSEESITEY β-casein ETIESLSSSEESITEYKQ β-casein ETIESLSSSEESITEYKQK β-casein 1.33 < IESLSSSEESITEYK β-casein IMEVPKAKDTVYT β-casein LLNPTHQIYPVTQPLAP β-casein < LLNPTHQIYPVTQPLAPVHNPIS β-casein < LLNPTHQIYPVTQPLAPVHNPISV β-casein LPIPQQVVPYP β-casein LPVPQPEIMEVPK β-casein

3 MEVPKAKDTVYTKG β-casein PLMQQVPQPIPQTL β-casein RETIESLSSSEESITEYK β-casein SEESITEYK β-casein SLSSSEESITE β-casein SLSSSEESITEY β-casein SLSSSEESITEYK β-casein 1.15 < SLSSSEESITEYKQKV β-casein SSSEESITEYK β-casein TEYKQKVEKVKHED β-casein < VLPIPQQVVPYPQR β-casein VPQPEIMEVPK β-casein 0.84 < IPQRQYLPNSHPPTVV κ-casein NEYNQLQLQAAHAQEQIR α-s1-casein Positive values indicate an upregulation and negative values indicate a downregulation in fold change peptide intensity. Bold peptide sequences were significant upregulated in both GA and LS groups.

4 Supplemental Table 3: Differing peptide sequences by GA, LS and the interaction of GA and LS Group Sequence Protein Group ANOVA p-value Preterm vs Term Gestational Age ETIESLSSSEESITEYKQ β-casein < ADGSRASVDSGSSEEQGGSSRA PIGR SEESITEYK β-casein PKAKDTVYT β-casein SLSSSEESITEYK β-casein SLSSSEESITE β-casein ADGSRASVDSGSSEEQGGSS PIGR LSSSEESITEYK β-casein ESLSSSEESITEYK β-casein SSSEESITEYKQKV β-casein ETIESLSSSEESITEYKQKV β-casein SSSEESITEYK β-casein AVADTRDQADGSRASVDSGSSEEQ PIGR SRASVDSGSSEEQGGSSRA PIGR ETIESLSSSEESITEYK β-casein AVADTRDQADGSRASVDSG PIGR ETIESLSSSEESITEYKQK β-casein RYPERLQNPSE α S1 -casein EESITEYKQKV β-casein GSRASVDSGSSEEQGGSSRA PIGR ETIESLSSSEESITEY β-casein NQELLLNPTHQIYPVT β-casein LSSSEESITEYKQKV β-casein SSEESITEYK β-casein EESITEYK β-casein DQADGSRASVDSGSSEEQG PIGR ADTRDQADGSRASVDSGSSEEQGGSS PIGR PQIPKLTD β-casein GAKTTFDVYTESWAQDPSQENK bile salt-activated lipase SLSSSEESITEYKQKV β-casein Early vs. Late Lactational Stage SLSSSEESITEYK β-casein < LLNPTHQIYPVTQPLAP β-casein < VPQPEIMEVPK β-casein < LLNPTHQIYPVTQPLAPVHNPIS β-casein < EESITEYK β-casein <

5 ETIESLSSSEESITEYKQK β-casein < LPIPQQVVPYP β-casein < SEESITEYK β-casein TEYKQKVEKVKHED β-casein ETIESLSSSEESITEY β-casein DSGSSEEQGGSSRALVSTLV PIGR SSSEESITEYK β-casein NEYNQLQLQAAHAQEQIR α S1 -casein DLENLHLP β-casein ESLSSSEESITEYK β-casein SLSSSEESITEYKQKV β-casein SLSSSEESITEY β-casein MEVPKAKDTVYTKG β-casein IMEVPKAKDTVYT β-casein LNPTHQIYPVTQ β-casein ETIESLSSSEESITEYKQ β-casein LPVPQPEIMEVPK β-casein ADTRDQADGSRASVD PIGR PLMQQVPQPIPQTL β-casein EDQQQGEDEHQDKIYPS β-casein LLNPTHQIYPVTQPLAPVHNPISV β-casein VDSGSSEEQGGSSRA PIGR AKDTVYTKGRVM β-casein VLPIPQQVVPYPQR β-casein IESLSSSEESITEYK β-casein TEYKQKVEKV β-casein SGSSEEQGGSSRAL PIGR ADTRDQADGSRASVDSGSSEEQGGSS PIGR LAQPAVVLPVPQPEIMEVPK β-casein NPTAHENYEKNNVML α S1 -casein ETIESLSSSEESITEYK β-casein AVADTRDQADGSRASVD PIGR IPQRQYLPNSHPPTVV κ-casein PLAPVHNPISV β-casein AVADTRDQADGSRA PIGR SLSSSEESITE β-casein Interaction of GA and LS SLSSSEESITEYK β-casein SEESITEYK β-casein LLNPTHQIYPVTQPLAPVHNPIS β-casein ETIESLSSSEESITEYKQ β-casein

6 ETIESLSSSEESITEYKQK β-casein LLNPTHQIYPVTQPLAP β-casein EESITEYK β-casein VPQPEIMEVPK β-casein SSSEESITEYK β-casein ESLSSSEESITEYK β-casein ETIESLSSSEESITEY β-casein DLENLHLP β-casein TEYKQKVEKVKHED β-casein LPIPQQVVPYP β-casein ADGSRASVDSGSSEEQGGSSRA PIGR SLSSSEESITE β-casein PKAKDTVYT β-casein DSGSSEEQGGSSRALVSTLV PIGR SLSSSEESITEYKQKV β-casein SLSSSEESITEY β-casein DQADGSRASVDSGSSEEQGGSSRALVSTL PIGR MEVPKAKDTVYTKG β-casein NEYNQLQLQAAHAQEQIR α S1 -casein ADGSRASVDSGSSEEQGGSS PIGR LNPTHQIYPVTQ β-casein ETIESLSSSEESITEYK β-casein ADTRDQADGSRASVD PIGR LSSSEESITEYK β-casein Bold peptide sequences were uniquely significant for the designated group, and were not found significant in other groups.

7 Supplemental Table 4: Prediction of the enzymes responsible for cleavage of human milk parent proteins, ranked by total number of cleavages Enzymes N- terminal cleavage count C- terminal cleavage count Total cleavages Unique cleavage Number of expected cleavages within the peptide Number of proteins cleaved Odds Ratio Standard Error trypsin 1 a plasmin cathepsin D LysC pepsin (ph 1.3) elastase modified chymotrypsin chymotrypsin low pepsin (ph > 2) Pepsin (ph 1.3) AspN endopeptidase argcproteinase clostripain proline endopeptidase diff pepsin (ph > 2) formic acid staphprotease glutamyl endopeptidase a staphylococcal peptidase I chymotrypsin high chymotrypsin low CNBr cyanogen bromide chymotrypsin low proline endopeptidase iodosobenzoic acid iodosobenzoate chymotrypsin low chymotrypsin high NTCB thrombin a Enzymes that do not have expression in milk, but data indicate the presence of other enzymes with similar activity

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9 Supplemental Figure 1: Panel A: Quantitative hierarchical k-means clustering shows samples as the column tree and peptide sequences as the row tree, with lower intensities plotted as dark blue with an increasing color scale from gray to dark red as the log10 fold change of the peptide intensity increases. Panel B shows the zoomed in portion of the top of Panel A where peptide intensities were highest, the row tree indicateds the partent protein of the most intense peptides.

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11 Supplemental Figure 2: Volcano plots depicting log 10 fold change in peptide intensity (x axis) and -log p value (yaxis). All significant peptides are represented as filled circles and non-significant peptides are unfilled. Panel A, peptide intensity distribution between preterm late LS (blue) and term early LS milk (red). Panel B, peptide intensity distribution between preterm late LS (blue) and term late LS milk (red). Panel C, peptide intensity distribution between term early LS (blue) and term late LS milk (red).