Top-down proteomics: Separation and mass spectral characterization of complex cell lysates from Methanosarcina acetivorans and human cancer cell

While most proteins are enzymatically digested (i.e., bottom-up) prior to analysis with a mass spectrometer, keeping a protein intact (i.e., top-down) provides an informatic advantage by allowing for concurrent analysis of all mass modifications. However, many of the tools that have been developed for the bottom-up strategy are not well-defined or are nonexistent for intact proteins. Top-down requires specialized tools to overcome inherent challenges in dealing with large proteins >5 kDa. One such tool is a mass spectrometer capable of offering high resolution data of large proteins. With the advent of high magnetic field Fourier transform ion cyclotron resonance mass spectrometers (FTICR-MS), obtaining high resolution data is routine. With a solid instrument for data acquisition, challenges still remain on improving protein chromatography and increasing throughput in data acquisition and database searching.;Chromatography, automated data acquisition, and bioinformatics represent the efforts pre-, during, and post-data acquisition on the mass spectrometer. Nano-LCMS/MS has been utilized for the quick analysis of HeLa S3 cancer cells and Methanosarcina acetivorans. Nano-LC-MS/MS has proven to be a quick and sensitive method for intact protein characterization. Starting with a 104 equivalent of HeLa S3 cells, a nano-LC-MS/MS analysis of a nuclei sulfuric acid extraction identified several intact proteins including calgizarrin, ubiquitin, ribosomal proteins, and histones. An analysis of M. acetivorans proteins by nano-LC-MS/MS identified several new proteins including a mercury ion binding protein with a 2 Da negative mass discrepancy due to a disulfide bond. Furthermore, several mis-predicted sites have been characterized where the start methionine has been incorrectly annotated, and four small unidentified reading frame ("smURF") proteins were discovered despite not being annotated during automated annotation of this archaeal genome.;Data acquisition for FTICR-MS is a time consuming process with transients requiring up to 8 s/scan. However, the improvement in spectral quality from averaging multiple scans can make offline analysis a required strategy. To maximize the benefit of an offline approach, an online-offline automation strategy is presented for the analysis of M. acetivorans and HeLa S3 cells. For HeLa S3 cells, this equated to the identifications of 51 intact protein forms including H2A.X, a protein notoriously difficult to characterize in an asynchronous cell lysate. For M. acetivorans this approach has led to the identification of a smURF protein with an unusual AUA start methionine.;The implementation of high-throughput platforms generated thousands of spectra that must be searched for protein identifications. To maximize the effectiveness of protein searching, an iterative search strategy is presented that makes use of multiple search modes in ProSight HT. Greater than 90% of all identified proteins were discovered in this manner.;The combination of nano-LC-MS/MS with an online-offline automation platform and an iterative data search strategy has allowed for high-throughput data acquisition to be possible with top-down protein analysis. Taken together, these platforms represent the next step in increasing the utility of top-down.