Optimization for peptide sample preparation for urine peptidomics

Tara K Sigdel,Carrie D Nicora,Wei-Jun Qian,Minnie M Sarwal,Szu-Chuan Hsieh,Hong Dai,David G Camp

doi:10.1186/1559-0275-11-7

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.

Highlights

Disease specific biomarkers remain as an unmet need in an overwhelming majority of cases for monitoring human health
Unlike urine peptide analysis by MALDI [17,18,19] urine peptidomics analysis by LC-MS is highly sensitive to the presence of unidentified contaminants that exist in urine, which if not effectively removed by further purification, impair the performance of the LC-MS analysis
When the standard solid phase extraction (SPE) [20] method of urine peptide extraction and purification was utilized for sample preparation, persisting contaminants in the urine, inclusive of urobilin and urobilinogen, clog the LC column and interfere with the assay performance (Sigdel et al, unpublished data) we report a mandatory modification of the SPE method, termed modified-SPE method, which provides optimal peptide extraction and purification for urinary peptide analysis by LC-MS

Summary

Introduction

Disease specific biomarkers remain as an unmet need in an overwhelming majority of cases for monitoring human health. Though there is an ever increasing effort to identify specific and sensitive biomarkers for monitoring human health and for the early detection of disease onset, many hurdles still exist in identifying effective biomarkers [1]. In this context, urine could prove to be an important proximal fluid in providing biomarkers that could be tested noninvasively [2]. The emergence of new and sophisticated methods of molecular profiling have aided in our ability to analyze peptides in complex biological mixtures in combination with the analysis of their degradation patterns This information may provide important clues about underlying (patho) physiological processes [11,12]. When the standard solid phase extraction (SPE) [20] method of urine peptide extraction and purification was utilized for sample preparation, persisting contaminants in the urine, inclusive of urobilin and urobilinogen, clog the LC column and interfere with the assay performance (Sigdel et al, unpublished data) we report a mandatory modification of the SPE method, termed modified-SPE (mSPE) method, which provides optimal peptide extraction and purification for urinary peptide analysis by LC-MS

Methods

Results

Conclusion