Stop-flow Two-dimensional Liquid Chromatography and Its Application in Separation and On-line Detection for Bioactive Peptides from Food-derived Protein Hydrolysates

Author:Xu Ju Cai

Supervisor:zhao mou ming


Degree Year:2019





Benefitting from the advantages in food safety,absorption and sources,the food-derived bioactive peptides have been the focus of food research in recent years.However,only a few bioactive peptides have been identified from the complex food-derived protein hydrolysates due to the limited efficiency in conventional separation and purification techniques.In this work,the additional band broadening of peptides during stop-flow operation in the first dimensional(1D)size-exclusion chromatography(SEC)and reversed phase chromatography(RPLC)were studied.Besides,the two-dimensional peak detection method was also improved with retention time and bidirectional overlap criteria added.In addition,a fast stop-flow SEC×RPLC tandem mass spectrometry(MS)was designed and constructed in house for improving peptide separation and identification.Moreover,an on-line stop-flow RPLC×SEC-MS/DPPH assay system was developed for rapid separation and identification of bioactive peptides.The main research contents and results are as follows:(1)A fully automated stop-flow SEC×RPLC system was designed and constructed in house.The effects of different stop-flow operational parameters on band broadening in the 1D SEC column were quantitatively evaluated.Results showed that the effects of peak parking position and the number of stop-flow periods on band broadening were relatively small.Unlike large molecules,additional band broadening was evidently observed for small molecules in long-time analysis due to the relatively high effective diffusion coefficient(Deff).Therefore,shorter analysis time and lower 1D column temperature were suggested for analyzing small molecules.The stop-flow two-dimensional liquid chromatography(2D-LC)was further tested on peanut peptides and an evidently improved resolution was observed for both stop-flow heart-cutting and comprehensive 2D-LC analysis.The stop-flow SEC×RPLC,especially heart-cutting analysis with shorter analysis time and higher 1D resolution for selected fractions,offers a promising approach for efficient analysis of complex samples.(2)To improve the peptide analysis in SEC,a stop-flow RPLC×SEC system was designed and constructed in house,and the effects of different stop-flow operational parameters on the additional band broadening were quantitatively evaluated.Unlike analytes of large molecular size or long retention time with low Deff,additional band broadening was evidently observed for small molecules of short retention time(high Deff).Therefore,optimal flow rate,low column temperature and short stop-flow time were suggested for analyzing small molecules of short retention time.The established stop-flow 2D-LC was further tested on different protein hydrolysates,and evidently improved resolution was obtained for both heart-cutting and comprehensive 2D-LC analysis.Compared with the heart-cutting analysis with higher 1D resolution for selective fractions,comprehensive analysis could provide more complete information about the molecular weight distribution of the eluting solutes along RPLC.(3)An improved algorithm was developed for 2D peak detection in complex 2D-LC data sets.To improve the peak detection with 2DLC analysis using shifting second dimension(2D)gradient gradients,variable thresholds were employed for the added retention time criterion.Besides,the bidirectional overlap criterion performed at specified height was recommended to improve detection for tailing peaks.The developed algorithm was further tested on data sets obtained from different 2DLC analysis for a complex peptide mixture,and evidently improved performance with accuracy rate over 60%was obtained by the algorithm,even in peak detection with 2DLC analysis under relatively low 1D sampling frequency or shifting 2D gradient gradients.The accuracy rate obtained by the developed algorithm was at least 6%higher than that by the Peters’method.Moreover,it was no longer necessary for the new method to perform 2D peak detection in different merging directions or with both directions considered.(4)To improve the peptidomic analysis for food-derived protein hydrolysates with conventional-scale liquid chromatography tandem mass spectrometry(LC-MS),a fast stop-flow two-dimensional liquid chromatography tandem mass spectrometry(2D-LC-MS)was designed and constructed in house.Through the chromatographic system optimization,the UPLC SEC column and HSS T3 column were found to be an appropriate column pair for two-dimensional separation,and the peak capacity reached as high as 1165,indicating high resolving power of the system for separating complex samples.While analyzing corn protein hydrolysates,the PSMs and MS2 obtained by the stop-flow 2D-LC-MS were at least 25%and100%higher than those by 1D-LC-MS,respectively.With application of the developed system in analysis of casein hydrolysates and soybean peptides,the number of detected 2D peaks,PSMs and MS2 were all over 200,9 k and 50 k,respectively.This fully demonstrated the utility of the stop-flow 2D-LC-MS in peptidomic analysis.Moreover,through search in Biopep database,6 casein-derived bioactive peptides were identified,as well as thousands of potential bioactive peptides.(5)A fully automated stop-flow RPLC×SEC-MS/DPPH assay system was designed and constructed in house for rapid separation and identification of antioxidant peptides.Through the optimization in 2D-SEC-DPPH screening method,optimal parameters were determined.The appropriate reaction coil length was 1000×0.25 mm i.d.,optimal DPPH·concentration was 200μM and the suitable reaction temperature was 40℃.Through the analysis of glutathione,the effectiveness of the developed stop-flow RPLC×SEC-MS/DPPH assay system in separation and identification of antioxidant peptides was verified.Furthermore,two dipeptides,GC and CW with strong DPPH free radical scavenging activity were successfully identified from analysis of a complex standard mixture.This fully demonstrated the utility,effectiveness and practicality of the developed system in separation and identification of peptides with DPPH free radical scavenging activity from complex food-derived protein hydrolysates.