Specifications

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Specifications

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Iodoacetamide Alkyne is a broad spectrum cysteine reactive probe that reacts with nucleophiles, such as cysteine in proteins and peptides to covalently bind cysteine residues to alkyne. The alkyne group can be used as a click-chemistry handle to attach a fluorophore or biotin to reactive cysteines in proteins for visualization by in-gel fluorescence or identification by mass spectrometry, respectively.

Specifications

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BSA Alkyne is commonly used positive control for click chemistry-based enrichment of alkyne-tagged proteins. Bovine Serum Albumin (BSA) is chemically modified on a single cysteine residue with N-Propargylmaleimide (MV 130.12).

Provided as lyophilized solid. To prepare a working 1 mg/ml solution, resuspend lyophilized BSA-conjugate in 500 µl of 0.5 x PBS pH 7.4. DO NOT ADD sodium azide.

Specifications

Peptide Structure

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IsoTaG H/L Biotin Alkyne probe takes advantage of the mass shift of stable isotope labels to enable mass-independent chemical proteomics platform that helps to address unique challenges of the proteome characterization. In this approach a unique isotopic signature is embedded exclusively into the peptides and it serves as a computationally recognizable full-scan MS reporter. A computational algorithm, termed isotopic signature transfer and mass pattern prediction (IsoStamp), for the detection of recoded species in full-scan mass spectra, was also developed by the Carolyn Bertozzi group. IsoTaG H/L Biotin Alkyne pack contains 2 mg of light [M] and heave [M+2] probe encoded by 2 C12 or 2 C13 atoms.

Specifications

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Extraordinary strength of the streptavidin-biotin interaction allows for efficient capturing of even highly dilute targets; however, it makes recovery of proteins from affinity resins challenging. Conventional methods to elute biotinylated proteins from immobilized avidin include the following: (i) denaturation of streptavidin by boiling the resin in a denaturing buffer that may include high concentrations of chaotropic salts, (ii) trypsin digestion of proteins while they are bound to the resin, or (iii) elution of proteins with excess free biotin. These protocols can co-elute contaminant proteins by releasing nonspecifically bound proteins and/or naturally biotinylated proteins concurrently with labeled proteins. In addition, some of these methods can cause elution of high levels of resin-based peptides along with the proteins of interest, resulting in further sample contamination.

Disulfide Biotin Alkyne probe eliminates a major limitation of the streptavidin-biotin affinity purification. This reagent contains a biotin moiety linked to an alkyne group through a spacer arm containing a cleavable disulfide linker. Captured biomolecules can be efficiently released under mild conditions (50 mM dithiothreitol, 10 mM 2-mercaptoethanol or 1% sodium borohydride) and the small molecular fragment (143.20 Da) left on the labeled protein following cleavage. These features make the cleavable probe especially attractive for use in biomolecular labeling and proteomic studies.

Specifications

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Description

While there has been much interest in profiling the intact glycoproteome, the complexity of glycoproteoforms (and more broadly, all proteoforms) remains challenging to completely define. Mass spectrometry (MS) is commonly employed for characterization of complex proteomic samples. A popular strategy for protein identification is the bottom-up shotgun proteomics approach. In this method, a mixture of proteins is subjected to proteolytic digestion, the resulting peptides are separated by LC and detected by MS, and their parent proteins are inferred from the assigned peptide sequences.

To convert MS data acquired from proteolytic digests into protein identifications, tandem MS can be used to obtain sequence information for individual peptides, followed by comparing an in-silico proteolytic digest of an organism’s proteome. Typically, only the most abundant peptides are selected for fragmentation (Figure 2), whereas data for those peptides in relatively low quantities are not obtained. An inherent problem in shotgun proteomics is identifying proteins of low abundance, such as biomarkers for disease states, against a background of proteins whose concentrations can span up to 12 orders of magnitude.

To address the unique challenges of the global characterization of the intact glycoproteome, a mass-independent chemical glycoproteomics platform, termed isotope targeted glycoproteomics (IsoTag) was developed by the Carolyn Bertozzi group. The platform is comprised of four central components: (i) metabolic labeling with a chemically functionalized glycan, (ii) chemical tagging and enrichment using an isotopic recoding affinity probe, (iii) directed tandem MS, and (iv) targeted glycopeptide assignment (Figure 2).

IsoTaG is performed by isotopic recoding and enrichment of metabolically labeled glycoproteins followed by directed tandem MS (MS2 or MSn) analysis and intact glycopeptide assignment. Isotopic recoding is accomplished by metabolic labeling of cell or tissue samples with azide- or alkyne-functionalized sugars, followed by chemical conjugation with a biotin probe bearing a unique isotopic signature.

Some examples of sugar labels are peracetylated N-azidoacetylmannosamine (Ac4ManNAz), which is converted to the corresponding azidosialic acid (SiaNAz), and peracetylated N-azidoacetylgalactosamine (Ac4GalNAz), which is metabolized to label glycans possessing N-acetylglucosamine (GlcNAc) or N-acetylgalactosamine (GalNAc) (not provided with kit).

In order to perform isotopic tagging, the kit provides two cleavable IsoTaG probes encoded by zero [M] and two [M + 2] deuterium atoms. Probes with different encoding can be provided by Click Chemistry Tools though custom synthesis. The IsoTaG probe with zero, and that with two deuterium atoms [M, M + 2] can be used in different proportions; 1:1, 1:2, 1:3 and 1:4. Pattern recognition with isotopic ratio of 1:3 showed the highest fidelity.

Through these probes, a unique isotopic signature is embedded exclusively into the glycopeptides. The isotopic signature serves as a computationally recognizable full-scan MS reporter. A computational algorithm, termed isotopic signature transfer and mass pattern prediction (IsoStamp), for the detection of recorded species in full-scan mass spectra, was also developed by the Carolyn Bertozzi group. IsoStamp compares observed and predicted isotopic envelopes to identify chemically tagged species in full-scan mass spectra.

IsoTag has the potential to enhance any proteomics platform that employs chemical labeling for targeted protein identification, including isotope-coded affinity tagging, isobaric tagging for relative and absolute quantitation, and chemical tagging strategies for post-translational modification.

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The sample pack contains 4 most often used alkyne-activated, cleavable biotin probes, Diazo, Dde, PC and DADPS Biotin Alkyne. These probes allow for efficient release of streptavidin-bound proteins under mild conditions. Even though all 4 cleavable moieties undergo efficient cleavage, the recovery of biopolymer or digests might vary from application to application requiring a preliminary testing of biotin probes to determine a best performing linker for a given application. Sample pack is a cost efficient option to profile all 4 cleavable linkers.

Specifications

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Extraordinary strength of the streptavidin-biotin interaction allows for efficient capturing of even highly dilute targets; however, it makes recovery of proteins from affinity resins challenging. Conventional methods to elute biotinylated proteins from immobilized avidin include the following: (i) denaturation of streptavidin by boiling the resin in a denaturing buffer that may include high concentrations of chaotropic salts, (ii) trypsin digestion of proteins while they are bound to the resin, or (iii) elution of proteins with excess free biotin. These protocols can co-elute contaminant proteins by releasing nonspecifically bound proteins and/or naturally biotinylated proteins concurrently with labeled proteins. In addition, some of these methods can cause elution of high levels of resin-based peptides along with the proteins of interest, resulting in further sample contamination.

DADPS (dialkoxydiphenylsilane) Biotin Alkyne probes eliminate a major limitation of the streptavidin-biotin affinity purification. This reagent contains a biotin moiety linked to an azide moiety through a spacer arm containing a cleavable DADPS linker. Captured biomolecules can be efficiently released under mild conditions (10% formic acid, 0.5 h) and the small (84 Da) molecular fragment left on the labeled protein following cleavage. These features make the DADPS probe especially attractive for use in biomolecular labeling and proteomic studies.

Specifications

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Extraordinary strength of the streptavidin-biotin interaction allows for efficient capturing of even highly dilute targets; however, it makes recovery of proteins from affinity resins challenging. Conventional methods to elute biotinylated proteins from immobilized avidin include the following: (i) denaturation of streptavidin by boiling the resin in a denaturing buffer that may include high concentrations of chaotropic salts, (ii) trypsin digestion of proteins while they are bound to the resin, or (iii) elution of proteins with excess free biotin. These protocols can co-elute contaminant proteins by releasing nonspecifically bound proteins and/or naturally biotinylated proteins concurrently with labeled proteins. In addition, some of these methods can cause elution of high levels of resin-based peptides along with the proteins of interest, resulting in further sample contamination.

Diazo Biotin-Azide probes eliminate a major limitation of the streptavidin-biotin affinity purification. This reagent contains a biotin moiety linked to an azide moiety through a spacer arm containing a cleavable linker. Captured biomolecules can be efficiently released under mild conditions (25 mM sodium dithionite) and the small (262.30 Da) molecular fragment left on the labeled protein following cleavage. These features make the Diazo probe especially attractive for use in biomolecular labeling and proteomic studies.

Specifications

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