Adding polyvinylpyrrolidone to low level protein samples significantly improves peptide recovery in FASP digests: An inexpensive and simple modification to the FASP protocol.

Filter-aided sample preparation (FASP) remains a popular choice for proteomic sample preparation, particularly for its ability to produce a 'clean' peptide sample clear of large molecule contaminants. However, sample loss continues to be a problem particularly for sample inputs that contain less than ten micrograms of protein. Here, we describe that the simple addition of a polymer, polyvinylpyrrolidone-40 (PVP-40) to the protein sample prior to FASP digest significantly improves peptide recovery and identifications, especially with lower level sample inputs. PVP-FASP produces clean samples which required no additional sample clean-up prior to nanoLC-MS analysis. In addition, PVP-FASP is compatible with other FASP modifications, including the use of sodium deoxycholate (DOC) to improve trypsin digestion. SIGNIFICANCE: Simple modification to FASP procedure improves sample recovery during proteomic digests in SDS, improving peptide identifications and median peptide intensity

Glycoproteomic analysis of two mouse mammary cell lines during transforming growth factor (TGF)-β induced epithelial to mesenchymal transition

<p>Abstract</p> <p>Background</p> <p>TGF-β acts as an antiproliferative factor in normal epithelial cells and at early stages of oncogenesis. However, later in tumor development TGF-β can become tumor promoting through mechanisms including the induction of epithelial-to-mesenchymal transition (EMT), a process that is thought to contribute to tumor progression, invasion and metastasis. To identify EMT-related breast cancer therapeutic targets and biomarkers, we have used two proteomic approaches to find proteins that change in abundance upon the induction of EMT by TGF-β in two mouse mammary epithelial cell lines, NMuMG and BRI-JM01.</p> <p>Results</p> <p>Preliminary experiments based on two-dimensional electrophoresis of a hydrophobic cell fraction identified only 5 differentially expressed proteins from BRI-JM01 cells. Since 3 of these proteins were glycoproteins, we next used the lectin, wheat germ agglutinin (WGA), to enrich for glycoproteins, followed by relative quantification of tryptic peptides using a label-free LC-MS based method. Using these approaches, we identified several proteins that are modulated during the EMT process, including cell adhesion molecules (several members of the Integrin family, Fibronectin, Activated leukocyte cell adhesion molecule, and Neural cell adhesion molecule 1) and regulators of cellular signaling (Tumor-associated calcium signal transducer 2, Basigin).</p> <p>Conclusion</p> <p>Interestingly, despite the fact that TGF-β induces similar EMT phenotypes in NMuMG and BRI-JM01 cells, the proteomic results for the two cell lines showed only minimal overlap. These differences likely result in part from the conservative cut-off values used to define differentially-expressed proteins in these experiments. Alternatively, it is possible that the two cell lines may use different mechanisms to achieve an EMT transition.</p

Cell surface profiling of cultured cells by direct hydrazide capture of oxidized glycoproteins

Glycoproteins are a particularly interesting subset of the cellular proteome as a high proportion of proteins present on the extracellular cell surface are glycosylated. These cell surface proteins are ideal targets for biologic drug therapies or for diagnostics tests. Here, we describe a modification of the well-described Cell Surface Capture (CSC) method for the selective isolation and identification of cell surface glycoproteins that contain N-linked carbohydrates. This modification, which we refer to as Direct Cell Surface Capture (D-CSC), is based on oxidation of cell surface glycans on intact cells, followed by direct conjugation of the oxidized oligosaccharides to a solid support using hydrazide chemistry, with no biotinylation step. As a proof-of-principle, we applied D-CSC to the analysis of cell surface membrane proteins of three adherent cancer cell lines (A549, OVCAR3, and U87MG) and compared our results to those published using the well-established Cell Surface Capture (CSC) method, demonstrating comparable selectivity for cell surface proteins. • A method enabling the identification of cell surface proteins from cells in culture is described. • Application of this method to profile the cell surface on three different cancer cell lines is included

Application of microfluidic devices to proteomics research: identification of trace-level protein digests and affinity capture of target peptides

This report describes an integrated and modular microsystem providing rapid analyses of trace-level tryptic digests for proteomics applications. This microsystem includes an autosampler, a microfabricated device comprising a large channel (2.4 \u3bcl total volume), an array of separation channels, together with a low dead volume enabling the interface to nanoelectrospray mass spectrometry. The large channel of this microfluidic device provides a convenient platform to integrate C18 reverse phase packing or other type of affinity media such as immobilized antibodies or immobilized metal affinity chromatography beads thus enabling affinity selection of target peptides prior to electrophoretic separation and mass spectrometry analyses on a quadrupole/time-of-flight instrument. Sequential injection, preconcentration, and separation of peptide standards and tryptic digests are achieved with a throughput of up to 12 samples/per h and a concentration detection limit of 3c5 nm (25 fmol on chip). Replicate injections of peptide mixtures indicated that reproducibility of migration time was 1.2\u20131.8%, whereas relative standard deviation ranging from 9.2 to 11.8% are observed on peak heights. The application of this device for trace-level protein identification is demonstrated for two-dimensional gel spots obtained from extracts of human prostatic cancer cells (LNCap) using both peptide mass-fingerprint data base searching and on-line tandem mass spectrometry. Enrichment of target peptides prior to mass spectral analyses is achieved using c-myc-specific antibodies immobilized on protein G-Sepharose beads and facilitates the identification of antigenic peptides spiked at a level of 20 ng/ml in human plasma. Affinity selection is also demonstrated for gel-isolated protein bands where tryptic phosphopeptides are captured on immobilized metal affinity chromatography beads and subsequently separated and characterized on this microfluidic system.NRC publication: Ye

An accurate TMT-based approach to quantify and model lysine susceptibility to conjugation via N-hydroxysuccinimide esters in a monoclonal antibody

Abstract Conjugation of small molecules to proteins through N-hydroxysuccinimide (NHS) esters results in a random distribution of small molecules on lysine residues and the protein N-terminus. While mass spectrometry methods have improved characterization of these protein conjugates, it remains a challenge to quantify the occupancy at individual sites of conjugation. Here, we present a method using Tandem Mass Tags (TMT) that enabled the accurate and sensitive quantification of occupancy at individual conjugation sites in the NIST monoclonal antibody. At conjugation levels relevant to antibody drug conjugates in the clinic, site occupancy data was obtained for 37 individual sites, with average site occupancy data across 2 adjacent lysines obtained for an additional 12 sites. Thus, altogether, a measure of site occupancy was obtained for 98% of the available primary amines. We further showed that removal of the Fc-glycan on the NIST mAb increased conjugation at two specific sites in the heavy chain, demonstrating the utility of this method to identify changes in the susceptibility of individual sites to conjugation. This improved site occupancy data allowed calibration of a bi-parametric linear model for predicting the susceptibility of individual lysines to conjugation from 3D-structure based on their solvent exposures and ionization constants. Trained against the experimental data for lysines from the Fab fragment, the model provided accurate predictions of occupancies at lysine sites from the Fc region and the protein N-terminus (R2 = 0.76). This predictive model will enable improved engineering of antibodies for optimal labeling with fluorophores, toxins, or crosslinkers

Method for isolation and molecular characterization of extracellular microvesicles released from brain endothelial cells

<p>Abstract</p> <p>Background</p> <p>In addition to possessing intracellular vesicles, eukaryotic cells also produce extracellular microvesicles, ranging from 50 to 1000 nm in diameter that are released or shed into the microenvironment under physiological and pathological conditions. These membranous extracellular organelles include both exosomes (originating from internal vesicles of endosomes) and ectosomes (originating from direct budding/shedding of plasma membranes). Extracellular microvesicles contain cell-specific collections of proteins, glycoproteins, lipids, nucleic acids and other molecules. These vesicles play important roles in intercellular communication by acting as carrier for essential cell-specific information to target cells. Endothelial cells in the brain form the blood–brain barrier, a specialized interface between the blood and the brain that tightly controls traffic of nutrients and macromolecules between two compartments and interacts closely with other cells forming the neurovascular unit. Therefore, brain endothelial cell extracellular microvesicles could potentially play important roles in ‘externalizing’ brain-specific biomarkers into the blood stream during pathological conditions, in transcytosis of blood-borne molecules into the brain, and in cell-cell communication within the neurovascular unit.</p> <p>Methods</p> <p>To study cell-specific molecular make-up and functions of brain endothelial cell exosomes, methods for isolation of extracellular microvesicles using mass spectrometry-compatible protocols and the characterization of their signature profiles using mass spectrometry -based proteomics were developed.</p> <p>Results</p> <p>A total of 1179 proteins were identified in the isolated extracellular microvesicles from brain endothelial cells. The microvesicles were validated by identification of almost 60 known markers, including Alix, TSG101 and the tetraspanin proteins CD81 and CD9. The surface proteins on isolated microvesicles could potentially interact with both primary astrocytes and cortical neurons, as cell-cell communication vesicles. Finally, brain endothelial cell extracellular microvesicles were shown to contain several receptors previously shown to carry macromolecules across the blood brain barrier, including transferrin receptor, insulin receptor, LRPs, LDL and TMEM30A.</p> <p>Conclusions</p> <p>The methods described here permit identification of the molecular signatures for brain endothelial cell-specific extracellular microvesicles under various biological conditions. In addition to being a potential source of useful biomarkers, these vesicles contain potentially novel receptors known for delivering molecules across the blood–brain barrier.</p

Glycoproteomic comparison of clinical triple-negative and luminal breast tumors

Triple-negative (TN) breast cancer accounts for 3c15% of breast cancers and is characterized by a high likelihood of relapse and a lack of targeted therapies. In contrast, luminal-type tumors that express the estrogen and progesterone receptors (ER+/PR+) and lack expression of human epidermal growth factor receptor 2 (Her2 12) are treated with targeted hormonal therapy and carry a better prognosis. To identify potential targets for the development of future therapeutics aimed specifically at TN breast cancers, we have used a hydrazide-based glycoproteomic workflow to compare protein expression in clinical tumors from nine TN (Her2\u2013/ER-/PR-) and nine luminal (Her2\u2013/ER+/PR+) patients. Using a label-free LC\u2013MS based approach, we identified and quantified 2264 proteins. Of these, 90 proteins were more highly expressed and 86 proteins were underexpressed in the TN tumors relative to the luminal tumors. The expression level of four of these potential targets was verified in the original set of tumors by Western blot and correlated well with our mass-spectrometry-based quantification. Furthermore, 30% of the proteins differentially expressed between luminal and TN tumors were validated in a larger cohort of 406 TN and 469 luminal tumors through corresponding differences in their mRNA expression in publically available microarray data. A group of 29 of these differentially expressed proteins was shown to correctly classify 88% of TN and luminal tumors using microarray data of their associated mRNA levels. Interestingly, even within a group of TN breast cancer patients, the expression levels of these same mRNAs were able to significantly predict patient survival, suggesting that these proteins play a role in the aggressiveness seen in TN tumors. This study provides a comprehensive list of potential targets for the development of diagnostic and therapeutic agents specifically aimed at treating TN breast cancer and demonstrates the utility of using publicly available microarray data to further prioritize potential targets.Peer reviewed: YesNRC publication: Ye

Flagellin and Outer Surface Proteins from Borrelia burgdorferi Are Not Glycosylated▿

We investigated the presence of glycoproteins in Borrelia burgdorferi. We did not find any evidence for glycosylation of the major outer membrane proteins OspA and OspB or the structural flagellar proteins FlaB and FlaA. We suggest that glycoproteins present on the surface of B. burgdorferi may be tightly bound culture medium glycoproteins