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For each digest, the initial rates were plotted against the log10 of the corresponding digest dilutions, yielding a sigmoidal curve; the standard curve was similarly processed.
All peptide fragments that were obtained for each digest were submitted to online protein database UniProt (UniProt Consortium) for searching.
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To determine whether arrays were arranged as expected, we performed 10 separate digests for each of the three predicted inserts; each digest contained a distinct restriction enzyme that was targeted to a specific segment within an array (Fig. 3).
For each restriction digest, 10 units of enzyme were used in a final volume of 10 µl and incubated at 37°C for 16 hr.
After washing under high stringency conditions, several fragments of variable intensity were detected for each endonuclease digest as showed in Figure 1A.
For each restriction digest, an equal amount of amplicon was subject to the same conditions without the addition of restriction enzyme.
For each PBM proteome digest sample, triplicate LC-nano-ESI-MSE datasets were acquired, The MSE data were then processed with ProteinLynx Global Server v2.3 (Waters) using default parameters.
For each sample set digest (pregnancy and non-pregnancy), on half (containing 100 μg of protein) was transfer to a fresh tube labeled with mTRAQ Reagent Δ8 for the global internal standard.
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