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Abstract
Shotgun proteomics has been broadly used for high-throughput analysis of proteins in biological systems. In shotgun proteomics, proteins are digested and the resulting peptides are analyzed using a combination of high performance liquid chromatography (HPLC) and mass spectrometry (MS). Our approach relies on accurate mass measurement of peptides by Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). In this work, we explore and develop methodologies to improve proteomic analysis. First, we develop an algorithm to automatically identify light/heavy peptide pairs and calculate peptide relative abundance ratios in 15N metabolic labeling data. This method provides over 99% accuracy in assigning peak pair and reduces the time of data analysis from over 100 hours to tens of minutes. In the second approach, we investigate the mass accuracy for higher mass peptides by using stored waveform inverse Fourier transform (SWIFT) excitation for MALDI-FTICR mass spectrometry. Analysis of measurement errors reveals that SWIFT excitation provides smaller deviations from stepwise-external calibration and better mass accuracy than chirp excitation for a wide mass range and for widely varying ion populations.A shotgun proteomics approach is presented for simultaneous identification and quantitation of the proteins from a proteome using accurate mass measurement and nitrogen stoichiometry. We demonstrate here the utilities of 15N-metabolic labeling for protein identification when using nitrogen stoichiometry as an additional search constraint and for protein quantitation from determining the intensity ratios of light/heavy peptide pairs. The combination of stepwise-external calibration and SWIFT excitation is applied and the mass measurement accuracy (MMA) is significantly improved. Last, we describe a novel calibration method, N15Cal, which corrects the space charge induced frequency shift in FTICR-MS analysis of 15N-metabolically labeled peptides from a batch digested proteome. N15Cal utilizes the information from the mass difference between the 14N/15N peptide peak pairs to correct for space charge induced mass shifts. There is no need to include an internal calibrant or to apply ion population control. N15Cal has been successfully applied to the LC-MALDI-FTICR data of 15N-metabolic labeling proteomics.