glycopeptide-capture-for-cell-surface-proteomics

Glycopeptide Capture for Cell Surface Proteomics

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, ...

Simon Fraser University

This Communication describes the photolysis of individual optically trapped vesicles for delivering spatially and temporally resolved stimuli to single cells. A submicrometer-sized vesicle, which encapsulates the stimuli of interest, is precisely positioned with respect to a select cell by optical trapping. Application of a single focused UV laser pulse photolyzes the trapped vesicle and releases the stimuli onto a localized region of the cell. The spatial resolution of this technique is on the order of several hundreds of nanometers, while the temporal resolution lies in the sub-microsecond range.

Spatially and temporally resolved delivery of stimuli to single cells.

This letter describes a new strategy for initiating a chemical reaction that is based on the laser-induced breakdown of a nanoscopic barrier, which physically separates the reactants in space. Because the breakdown of the barrier is fast ( approximately 0.3 micros) and owing to the nanometer dimension of the barrier, the reactants can be brought together and the reaction can be initiated rapidly. The time scale most suited for this method (from microseconds to tens of milliseconds) bridges nicely between the faster time scales that are accessible mostly with laser-based triggering experiments and the slower time scales that are studied most frequently with flow-based devices.

Fast initiation of chemical reactions with laser-induced breakdown of a nanoscale partition.

This paper describes a new method to measure the encapsulation efficiency of individual lipid vesicles. Single vesicles were first optically trapped (with a CW Nd:YAG laser at 1064 nm) and then photolyzed with a single 3-ns UV laser pulse (from a N(2) laser at 337 nm) to release the molecules encapsulated within the vesicle; confocal detection with single-molecule sensitivity (laser excitation at 488 nm from a CW Ar(+) laser) was used to measure the number of released molecules. By placing the confocal probe volume a few micrometers from the vesicle and by monitoring the transit times and the number of released molecules that crossed this probe volume, we could calculate the total number of molecules released from the vesicle using a three-dimensional diffusion equation. Unlike traditional definitions of encapsulation efficiencies based on bulk assays, because we can measure the contents of and release from individual vesicles, we can define the encapsulation efficiency by dividing the concentration of molecules encapsulated in each vesicle over the original concentration of the molecules present in the loading solution. We characterized the encapsulation efficiency of carboxyfluorescein for vesicles prepared by rotary evaporation and found oligolamellar vesicles have an encapsulation efficiency of 36.3 +/- 18.9%, while multilamellar vesicles have an encapsulation efficiency of 17.5 +/- 8.9%.

Determination of the encapsulation efficiency of individual vesicles using single-vesicle photolysis and confocal single-molecule detection.

This paper describes the synthesis and loading of silica and polystyrene-acrylic based nanocapsules with small molecules. The nanocapsules are used for delivering defined packages of stimuli to single cells with both high spatial and temporal resolutions. To introduce molecules into the capsules, we characterized two approaches. The first approach is based on a base-swell process in which the shell of the capsule is swelled so small molecules can diffuse into the interior of the capsule and be trapped inside once the capsules are de-swelled. The second approach is based on a dry-swell-dry process in which the solution containing the molecules of interest and the nanocapsules is physically dried to promote more molecules to enter into the interior of the capsule. We characterized both methods by monitoring the content of and the release from individual capsules with confocal microscopy and wide-field imaging. To illustrate the biological applications of such nanocapsules, we used optical trapping to position a single carbachol-loaded capsule adjacent to a single CHO cell that has been transfected with muscarinic acetylcholine (M1) receptors, released the carbachol from the capsule with a single 3-ns N2 laser pulse, and then monitored the subsequent intracellular signaling triggered by the binding of carbachol to the M1 receptors.

Synthesis, loading, and application of individual nanocapsules for probing single-cell signaling.

This paper demonstrates the ability to grow silica directly on a deposited surface of polyelectrolyte. Using this strategy, we describe the deposition of layered polyelectrolyte-silica coating on negatively charged surfaces of polystyrene particles and latex nanocapsules, which could not be coated directly with silica alone. By etching away the underlying polystyrene bead, we were able to form polyelectrolyte-silica capsules that were mechanically robust. Using scanning and transmission electron microscopy, we imaged and studied the coating after the deposition of each layer of polyelectrolyte and silica. We then applied this new coating to latex nanocapsules that were loaded with fluorescein molecules. We found that the coating procedure did not cause the loaded molecules to leak out from the capsules, and we determined that the variation in the number of loaded molecules among capsules arose from differences in the volume of the nanocavities and was not caused by the loading and coating of the capsules. This layered architecture permits the thickness of the coating to be controlled in principle over a wide dynamic range, but more importantly, this coating could act as an effective seal to prevent undesired leakage from nanocapsules and thus increase the long-term storability of loaded capsules. Over a 30-day period, we determined that leakage from uncoated capsules was significant but negligible for ones that were coated with two layers of polyelectrolyte-silica. Using single-pulse UV photolysis of individual nanocapsules, we demonstrate that the molecules contained within coated capsules could be released effectively and on demand with a single laser pulse.

Layered polyelectrolyte-silica coating for nanocapsules.

We present a robust and general shotgun glycoproteomics approach to comprehensively profile glycoproteins in complex biological mixtures. In this approach, glycopeptides derived from glycoproteins are enriched by selective capture onto a solid support using hydrazide chemistry followed by enzymatic release of the peptides and subsequent analysis by tandem mass spectrometry. The approach was validated using standard protein mixtures that resulted in a close to 100% capture efficiency. Our capture approach was then applied to microsomal fractions of the cisplatin-resistant ovarian cancer cell line IGROV-1/CP. With a Protein Prophet probability value greater than 0.9, we identified a total of 302 proteins with an average protein identification rate of 136 +/- 19 (n = 4) in a single linear quadrupole ion trap (LTQ) mass spectrometer nano-LC-MS experiment and a selectivity of 91 +/- 1.6% (n = 4) for the N-linked glycoconsensus sequence. Our method has several advantages. 1) Digestion of proteins initially into peptides improves the solubility of large membrane proteins and exposes all of the glycosylation sites to ensure equal accessibility to capture reagents. 2) Capturing glycosylated peptides can effectively reduce sample complexity and at the same time increase the confidence of MS-based protein identifications (more potential peptide identifications per protein). 3) The utility of sodium sulfite as a quencher in our capture approach to replace the solid phase extraction step in an earlier glycoprotein chemical capture approach for removing excess sodium periodate allows the overall capture procedure to be completed in a single vessel. This improvement minimizes sample loss, increases sensitivity, and makes our protocol amenable for high throughput implementation, a feature that is essential for biomarker identification and validation of a large number of clinical samples. 4) The approach is demonstrated here on the analysis of N-linked glycopeptides; however, it can be applied equally well to O-glycoprotein analysis.

Shotgun glycopeptide capture approach coupled with mass spectrometry for comprehensive glycoproteomics.

Stem cells, with their profound implication in development and enormous potential in regenerative medicine, have been the subject of extensive molecular profiling studies in search of better markers and regulatory schema governing self-renewal versus differentiation. In this review article, we will discuss current advancement in high throughput technologies dedicated to the transcriptome, proteome and genome-wide localization analyses, and how they have been adopted in molecular profiling of stem cells with an emphasis on embryonic stem cell (ESC), hematopoietic stem cell (HSC) and neural stem cell (NSC).

Molecular profiling of stem cells.

E14.Tg2a mouse embryonic stem (mES) cells are a widely used host in gene trap and gene targeting techniques. Molecular characterization of host cells will provide background information for a better understanding of functions of the knockout genes. Using a highly selective glycopeptide-capture approach but ordinary liquid chromatography coupled mass spectrometry (LC-MS), we characterized the N-glycoproteins of E14.Tg2a cells and analyzed the close relationship between the obtained N-glycoproteome and cell-surface proteomes. Our results provide a global view of cell surface protein molecular properties, in which receptors seem to be much more diverse but lower in abundance than transporters on average. In addition, our results provide a systematic view of the E14.Tg2a N-glycosylation, from which we discovered some striking patterns, including an evolutionarily preserved and maybe functionally selected complementarity between N-glycosylation and the transmembrane structure in protein sequences. We also observed an environmentally influenced N-glycosylation pattern among glycoenzymes and extracellular matrix proteins. We hope that the acquired information enhances our molecular understanding of mES E14.Tg2a as well as the biological roles played by N-glycosylation in cell biology in general.

N-glycoproteome of E14.Tg2a mouse embryonic stem cells.

Blood is an ideal window for viewing our health and disease status. Because blood circulates throughout the entire body and carries secreted, shed, and excreted signature proteins from every organ and tissue type, it is thus possible to use the blood proteome to achieve a comprehensive assessment of multiple-organ physiology and pathology. To date, the blood proteome has been frequently examined for diseases of individual organs; studies on compound insults impacting multiple organs are, however, elusive. We believe that a characterization of peripheral blood for organ-specific proteins affords a powerful strategy to allow early detection, staging, and monitoring of diseases and their treatments at a whole-body level. In this paper we test this hypothesis by examining a mouse model of acetaminophen (APAP)-induced hepatic and extra-hepatic toxicity. We used a glycocapture-assisted global quantitative proteomics (gagQP) approach to study serum proteins and validated our results using Western blot. We discovered in mouse sera both hepatic and extra-hepatic organ-specific proteins. From our validation, it was determined that selected organ-specific proteins had changed their blood concentration during the course of toxicity development and recovery. Interestingly, the peak responding time of proteins specific to different organs varied in a time-course study. The collected molecular information shed light on a complex, dynamic, yet interweaving, multiorgan-enrolled APAP toxicity. The developed technique as well as the identified protein markers is translational to human studies. We hope our work can broaden the utility of blood proteomics in diagnosis and research of the whole-body response to pathogenic cues.

Glycocapture-assisted global quantitative proteomics (gagQP) reveals multiorgan responses in serum toxicoproteome.

The advent of proteomics technology has transformed our understanding of biological membranes. The challenges for studying membrane proteins have inspired the development of many analytical and bioanalytical tools, and the techniques of glycoproteomics have emerged as an effective means to enrich and characterize membrane and plasma-membrane proteomes. This Review summarizes the development of various glycoproteomics techniques to overcome the hurdles formed by the unique structures and behaviors of membrane proteins with a focus on N-glycoproteomics. Example contributions of N-glycoproteomics to the understanding of membrane biology are provided, and the areas that require future technical breakthroughs are discussed.

Article	Year
Spatially and temporally resolved delivery of stimuli to single cells. Journal of the American Chemical Society\| PubMed ID: 12656592	2003
Fast initiation of chemical reactions with laser-induced breakdown of a nanoscale partition. Langmuir : the ACS journal of surfaces and colloids\| PubMed ID: 15491172	2004
Determination of the encapsulation efficiency of individual vesicles using single-vesicle photolysis and confocal single-molecule detection. Analytical chemistry\| PubMed ID: 15859592	2005
Synthesis, loading, and application of individual nanocapsules for probing single-cell signaling. Langmuir : the ACS journal of surfaces and colloids\| PubMed ID: 15969173	2004
Layered polyelectrolyte-silica coating for nanocapsules. Langmuir : the ACS journal of surfaces and colloids\| PubMed ID: 16262349	2005
Shotgun glycopeptide capture approach coupled with mass spectrometry for comprehensive glycoproteomics. Molecular & cellular proteomics : MCP\| PubMed ID: 17074749	2007
Molecular profiling of stem cells. Clinica chimica acta; international journal of clinical chemistry\| PubMed ID: 17266947	2007
N-glycoproteome of E14.Tg2a mouse embryonic stem cells. PloS one\| PubMed ID: 23405203	2013
Glycocapture-assisted global quantitative proteomics (gagQP) reveals multiorgan responses in serum toxicoproteome. Journal of proteome research\| PubMed ID: 23540550	2013
Protein-Centric N-Glycoproteomics Analysis of Membrane and Plasma Membrane Proteins. Journal of proteome research\| PubMed ID: 24754784	2014

Bingyun Sun

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