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Optimization for peptide sample preparation for urine peptidomics.

ABSTRACT: Analysis of native or endogenous peptides in biofluids can provide valuable insights into disease mechanisms. Furthermore, the detected peptides may also have utility as potential biomarkers for non-invasive monitoring of human diseases. The non-invasive nature of urine collection and the abundance of peptides in the urine makes analysis by high-throughput 'peptidomics' methods , an attractive approach for investigating the pathogenesis of renal disease. However, urine peptidomics methodologies can be problematic with regards to difficulties associated with sample preparation. The urine matrix can provide significant background interference in making the analytical measurements that it hampers both the identification of peptides and the depth of the peptidomics read when utilizing LC-MS based peptidome analysis. We report on a novel adaptation of the standard solid phase extraction (SPE) method to a modified SPE (mSPE) approach for improved peptide yield and analysis sensitivity with LC-MS based peptidomics in terms of time, cost, clogging of the LC-MS column, peptide yield, peptide quality, and number of peptides identified by each method. Expense and time requirements were comparable for both SPE and mSPE, but more interfering contaminants from the urine matrix were evident in the SPE preparations (e.g., clogging of the LC-MS columns, yellowish background coloration of prepared samples due to retained urobilin, lower peptide yields) when compared to the mSPE method. When we compared data from technical replicates of 4 runs, the mSPE method provided significantly improved efficiencies for the preparation of samples from urine (e.g., mSPE peptide identification 82% versus 18% with SPE; p = 8.92E-05). Additionally, peptide identifications, when applying the mSPE method, highlighted the biology of differential activation of urine peptidases during acute renal transplant rejection with distinct laddering of specific peptides, which was obscured for most proteins when utilizing the conventional SPE method. In conclusion, the mSPE method was found to be superior to the conventional, standard SPE method for urine peptide sample preparation when applying LC-MS peptidomics analysis due to the optimized sample clean up that provided improved experimental inference from the confidently identified peptides.

SUBMITTER: Sigdel TK

PROVIDER: S-EPMC3944950 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Bordetella pertussis, the aetiological agent of whooping cough, is re-emerging globally despite widespread vaccination. B. pertussis is highly infectious and, prior to vaccination programmes, was the leading cause of infant mortality. The WHO estimated that over 600?000 deaths are prevented annually by pertussis vaccination, but B. pertussis infection was still responsible for over 63?000 deaths globally in 2013. The re-emergence of B. pertussis has been linked to strains with inactive or absent major virulence factors included in vaccines such as pertactin, pertussis toxin and filamentous haemagglutinin. Thus, the molecular surveillance of currently circulating strains is critical in understanding and controlling B. pertussis. Such information provides data on strains to inform control measures and the identification of future vaccine antigens. Current surveillance and typing methods for B. pertussis rely on the availability of clinical isolates. However, since the 1990s, the majority of pertussis cases have been diagnosed by PCR, where an isolate is not needed. The rapid decline in the availability of B. pertussis isolates impacts our ability to monitor this infection. The growing uptake of next-generation sequencing (NGS) has offered the opportunity for culture-independent genome sequencing and typing of this fastidious pathogen. Therefore, the objective of the study was to optimize respiratory sample preparation, independent of culture, in order to type B. pertussis using NGS. The study compared commercial depletion kits and specimen-processing methods using selective lysis detergents. The goal was to deplete human DNA, the major obstacle for sequencing a pathogen directly from a clinical sample. Samples spiked with a clinically relevant amount of B. pertussis were used to provide comparison between the different methods. Commercial depletion kits including the MolYsis, Qiagen Microbiome and NEBNext Kits were tested. Previously published methods, for Saponin and TritonX-100, were also trialled as a depletion. The ratio of B. pertussis to human DNA was determined by real-time PCR for ERV3 and IS481 (as markers of human and B. pertussis DNA, respectively), then samples were sequenced using the Illumina NextSeq 500 platform. The number of human and B. pertussis sequenced reads were then compared between treatments. The results showed that commercial kits reduced the human DNA present, but also reduced the concentration of target B. pertussis. However, selective lysis with Saponin treatment resulted in almost undetectable levels of human DNA, with minimal loss of target B. pertussis DNA. Sequencing read depth improved five-fold in reads to B. pertussis. Our investigation delivered a potential protocol that will enable the public health laboratory surveillance of B. pertussis in the era of culture-independent testing.

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Optimization for peptide sample preparation for urine peptidomics.

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