Roland Luethy

Towards defining the urinary proteome using liquid chromatography‐tandem mass spectrometry I.Profiling an unfractionated tryptic digest

The proteome of normal male urine from a commercial pooled source has been examined using direct liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). The entire urinary protein mixture was denatured, reduced and enzymatically digested prior to LC‐MS/MS analysis using a hybrid‐quadrupole time‐of‐flight mass spectrometer (Q‐TOF) to perform data‐dependent ion selection and fragmentation. To fragment as many peptides as possible, the mixture was analyzed four separate times, with the mass spectrometer selecting ions for fragmentation from a subset of the entire mass range for each run. This approach requires only an autosampler on the HPLC for automation ( i.e, unattended operation). Across these four analyses, 1.450 peptide MS/MS spectra were matched to 751 sequences to identify 124 gene products (proteins and translations of expressed sequence tags). Interestingly, the experimental time for these analyses was less than that required to run a single two‐dimensional gel.

Automated LC-LC-MS-MS platform using binary ion-exchange and gradient reversed-phase chromatography for improved proteomic analyses.

A simple multidimensional liquid chromatography system utilizing an isocratic pump and a HPLC system is described for the comprehensive proteomic analysis of complex peptide digest mixtures by coupled LC-LC-MS-MS techniques. A binary ion-exchange separation was achieved through the use of a strong cation-exchange column followed by a reversed-phase column for data-dependent LC-MS-MS analysis of the unbound analytes, and following salt elution (and concomitant column reequilibration), the bound analytes. Off-line validation of the platform showed near quantitative recovery of fractionated peptides and essentially complete ion-exchange partitioning. In comparative analyses of a highly complex peptide digest mixture a >40% increase in the number of peptide and protein identifications was achieved using this multidimensional platform compared to an unfractionated control.

Towards defining the urinary proteome using liquid chromatography-tandem mass spectrometry. II. Limitations of complex mixture analyses.

With an emphasis on obtaining a multitude of high quality tandem mass spectrometry spectra for protein identification, instrumental parameters are described for the liquid chromatography‐tandem mass spectrometry analysis of trypsin digested unfractionated urine using a hybrid quadrupole‐time‐of‐flight (Q‐TOF) mass spectrometer. Precursor acquisition rates of up to 20 distinct precursors/minute in a single analysis were obtained through the use of parallel precursor selection (four precursors/survey period) and variable collision induced dissociation integration time (1 to 6 periods summed). Maximal exploitation of the gas phase fractionated ions was obtained through the use of narrow survey scans and iterative data‐dependent analyses incorporating dynamic exclusion. The impact on data fidelity as a product of data‐dependent selection of precursor ions from a dynamically excluded field is discussed with regards to sample complexity, precursor selection rates, survey scan range and facile chemical modifications. Operational and post‐analysis strategies are presented to restore data confidence and reconcile the greatest number of matched spectra.