Data-dependent precursor ion selection is widely used in shotgun proteomics to profile the protein components of complex samples. Although very popular, this bottom-up method presents major drawbacks in terms of detectable dynamic range. Here, we demonstrate the superior performance of a data-independent method we term Precursor Acquisition Independent From Ion Count (PAcIFIC). Our results show that almost the entire, predicted, soluble bacterial proteome can be thoroughly analyzed by PAcIFIC without the need for any sample fractionation other than the C18-based liquid-chromatograph used to introduce the peptide mixture into the mass spectrometer. Importantly, we also show that PAcIFIC provides unique performance for analysis of human plasma in terms of number of proteins identified (746 at FDR ≤ 0.5%) and achieved dynamic range (eight orders of magnitude at FDR ≤ 0.5%), without any fractionation other than immuno-depletion of the seven most abundant proteins.

High-density lipoprotein (HDL), a lipid nanoparticle containing many different low abundance proteins, is an attractive target for clinical proteomics because its compositional heterogeneity is linked to its cardioprotective effects. Selected reaction monitoring (SRM) is currently the method of choice for targeted quantification of proteins in such a complex biological matrix. However, model system studies suggest that parallel reaction monitoring (PRM) is more specific than SRM because many product ions can be used to confirm the identity of a peptide. We therefore compared PRM and SRM for their abilities to quantify proteins in HDL, using 15N-labeled apolipoprotein A-I (HDL’s most abundant protein) as the internal standard. PRM and SRM exhibited comparable linearity, dynamic range, precision, and repeatability for protein quantification of HDL. Moreover, the single internal standard protein performed as well as protein-specific peptide internal standards when quantifying 3 different proteins. Importantly, PRM and SRM yielded virtually identical quantitative results for 26 proteins in HDL isolated from 44 subjects. Because PRM requires less method development than SRM and is potentially more specific, our observations indicate that PRM in concert with a single isotope-labeled protein is a promising new strategy for quantifying HDL proteins in translational studies.

The identification and characterization of previously unidentified signal transduction molecules has expanded our understanding of biological systems and facilitated the development of mechanism-based therapeutics. We present a highly validated small interfering RNA (siRNA) screen that functionally annotates the human genome for modulation of the Wnt/beta-catenin signal transduction pathway. Merging these functional data with an extensive Wnt/beta-catenin protein interaction network produces an integrated physical and functional map of the pathway. The power of this approach is illustrated by the positioning of siRNA screen hits into discrete physical complexes of proteins. Similarly, this approach allows one to filter discoveries made through protein-protein interaction screens for functional contribution to the phenotype of interest. Using this methodology, we characterized AGGF1 as a nuclear chromatin-associated protein that participates in beta-catenin-mediated transcription in human colon cancer cells.

A by-product in the processing of prostate tissue for cell sorting by collagenase digestion is the media supernatant that remains after the cells are harvested. These supernatants contain proteins made by the cells within the tissue. Quantitative proteomic analysis of N-glycosylated proteins detected an increased amount of CD90/THY1 in cancer supernatants compared to non-cancer supernatants. Immunohistochemistry showed that in all carcinomas, regardless of Gleason grade, a layer of CD90-positive stromal fibroblastic cells, approximately 5-to-10 cells deep, was localized to tumor glands. In contrast, a no more than 1-cell wide girth of CD90-positive stromal cells was found around benign glands. The increased number of CD90-positive stromal cells in cancer correlated with overexpression of CD90 mRNA detected by gene expression analysis of stromal cells obtained by laser-capture microdissection. There is increasing evidence that cancer-associated stroma plays a role in both tumor progression and carcinogenesis. Most experiments to identify cancer biomarkers have focused on the cancer cells. CD90, being a marker for prostate cancer-associated stroma, might be a potential biomarker for this cancer. A non-invasive test could be provided by a urine test. Proteomic analysis of urine from patients with prostate cancer identified CD90; conversely, CD90 was not detected in the urine of post-prostatectomy patients. Furthermore, this urinary CD90 protein was a variant CD90 protein not known to be expressed by such cells as lymphocytes that express CD90. These CD90 results were obtained from ∼90 cases consisting of proteomic analysis of tissue and urine, immunohistochemistry, Western blot analysis of tissue media, flow cytometry of cells from digested tissue, and reverse transcriptase polymerase chain reaction analysis of isolated stromal cells.

In this paper, we present the results of proof-of-concept experiments using a novel photocleavable and mass spectrometry identifiable cross-linker pcPIR (photocleavable Protein Interaction Reporter). pcPIR can be dissociated under UV irradiation either off-or on-line before the introduction to the mass spectrometers. Photo dissociation of cross-linkers is different from either the gas phase or the chemical cleavage of cross-linkers. Different types of cross-links can be identified using the pcPIR mass relationships, where the mass of cross-linked precursor equals the sum of the masses of the released products and reporter. Since pcPIR is cleaved prior to the entrance to the mass spectrometer, the released peptides are available to be sequenced with routine CID MS/MS experiments and database search algorithms. In this report, the pcPIR strategy of identifying the cross-linked peptides with on-and off-line photocleavage coupled with novel targeted data dependent LC-MS/MS is demonstrated with the use of standard peptides, BSA and human hemoglobin tetramer protein complex.

The identification of peptides by microcapillary liquid chromatography-tandem mass spectrometry (µLC-MS/MS) has become routine because of the development of fast scanning mass spectrometers, data-dependent acquisition, and database searching algorithms. However, many peptides within the detection limit of the mass spectrometer remain unidentified because of limitations in MS/MS sampling speed despite the dynamic range and peak capacity of the instrument. We have developed an automated approach that uses the mass spectra from high resolution µLC-MS data to define the molecular species present in the mixture and directs the acquisition of MS/MS spectra to precursors that were missed in prior analyses. This approach increases the coverage of the molecular species sampled by MS/MS and consequently the number of peptides and proteins identified during the acquisition of technical or biological replicates using a simple one-dimensional chromatographic separation. The combination of a unique workflow and custom software contribute to the improved identification of molecular features detected in proteomics experiments of complex protein mixtures.

Plasma membranes of most cell types are thought to contain microdomains commonly referred to as lipid rafts, biochemically distinct from bulk plasma membrane and apparently enriched for proteins involved in signal transduction. In T cells, it is believed that lipid rafts aggregate at the site of T cell receptor engagement and act as foci for initiation of the signaling process. In order to gain insight into the possible functioning of lipid rafts, we applied microcapillary liquid chromatography-electrospray ionization-tandem mass spectrometry (µLC-ESI-MS/MS) methodologies to the identification of proteins which co-purified with lipid rafts. Following isolation of lipid rafts as Triton-insoluble, low-density membrane fractions from Jurkat T cells, tryptic digests were generated of electrophoretically-resolved, individual protein bands. Alternatively, avidin-affinity purification was used to isolate cysteine-containing peptides from total tryptic digests of unseparated lipid raft proteins following protein labeling with a cysteine-specific biotinylation reagent. In both cases, protein identifications were made by comparison of tandem mass spectra, generated by µLC-ESI-MS/MS, with both protein and DNA sequence databases using Sequest software. Proteins identified essentially fell into two groups: cytoskeletal proteins and proteins involved in signal transduction. These findings are discussed in light of the current understanding of both lipid-raft biology and signal transduction.

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