Global Analysis of the Cortical Neuron Proteome

Abstract

In this study, a multidimensional fractionation approach was combined with MS/MS to increase the capability of characterizing complex protein profiles of mammalian neuronal cells. Proteins extracted from primary cultures of cortical neurons were digested with trypsin followed by fractionation using strong cation exchange chromatography. Each of these fractions was analyzed by microcapillary reversed-phase LC-MS/MS. The analysis of the MS/MS data resulted in the identification of over 15,000 unique peptides from which 3,590 unique proteins were identified based on protein-specific peptide tags that are unique to a single protein in the searched database. In addition, 952 protein clusters were identified using cluster analysis of the proteins identified by the peptides not unique to a single protein. This identification revealed that a minimum of 4,542 proteins could be identified from this experiment, representing ϳ16% of all known mouse proteins. An evaluation of the number of false-positive identifications was undertaken by searching the entire MS/MS dataset against a database containing the sequences of over 12,000 proteins from archaea. This analysis allowed a systematic determination of the level of confidence in the identification of peptides as a function of SEQUEST cross correlation (X corr ) and delta correlation (⌬Cn) scores. Correlation charts were also constructed to show the number of unique peptides identified for proteins from specific classes. The results show that low-abundance proteins involved in signal transduction and transcription are generally identified by fewer peptides than high-abundance proteins that play a role in maintaining mammalian cellular structure and motility. The results presented here provide the broadest proteome coverage for a mammalian cell to date and show that MS-based proteomics has the potential to provide high coverage of the proteins expressed within a cell.

Proteomic Mapping of Brain Plasma Membrane Proteins

“…In the current study, off-line LC fractionation of endopeptidase Lys-C peptides was followed by trypsin digestion and LC-MS/MS analysis. Commonly employed 2D LC-MS/MS technology with low-resolution ion trap mass spectrometers (27)(28)(29)(30) frequently introduces low-confidence database hits and results in difficulties in data interpretation. In contrast, we employed either a Q-TOF or a linear ion trap FT mass spectrometer.…”

Membrane Proteomic Analysis of Arabidopsis thaliana Using Alternative Solubilization Techniques

“…Peptides were identified by searching the product ion spectra against the Arabidopsis thaliana database using TurboSEQUEST (BioWorks 3.1, Thermo Electron, San Jose, CA). Only tryptic peptides displaying a charge dependent cross-correlation score (Xcorr) of 2.1, 2.2, 2.5 and 2.9, for +1, +2 of <1200 u, +2 of g1200 u, and +3 charged species of the precursor ions and a delta-correlation score (∆C n) of at least 0.08 were used for identification as previously reported, 33 which are similar to other thresholds used for ion trap data. 34 All searches included a static carboxamidomethyl modification of 57.0 u on Cys residues due to alkylation and a differential phosphorylation modification of 79.98 u on Ser, Thr, and Tyr residues.…”

Quantitative analysis of the low molecular weight serum proteome using 18O stable isotope labeling in a lung tumor xenograft mouse model

“…MudPIT, or variations thereof, has been used in countless studies to characterize complex proteome samples, with the typical goal of obtaining as much possible information concerning the sample of interest. This technology has ushered in an era where upwards of 4000 proteins can be characterized in a single sample [6,7] and enables proteomics with the technology to live up to its expectations of comprehensive coverage of cellular proteins.…”