Protein Analysis and Purification

Purify and Conjugate Antibodies in a Single Workflow

Posted on by Gary Kobs

Isoform_Antibodies_LinkedInAntibodies labeled with small molecules such as fluorophore, biotin or drugs play a critical role in various areas of biological research,drug discovery and diagnostics. There are several limitations to current methods for labeling antibodies including the need for purified antibodies at high concentrations and multiple buffer exchange steps.

In a recent publication, a method (on-bead conjugation) is described that addresses these limitations by combining antibody purification and conjugation in a single workflow. This method uses high capacity-magnetic Protein A or Protein G beads to capture antibodies directly from cell media followed by conjugation with small molecules and elution of conjugated antibodies from the beads.

Using a variety of fluorophores the researchers show that the on-bead conjugation method is compatible with both thiol- and amine-based chemistry.

This method enables simple and rapid processing of multiple samples in parallel with high-efficiency antibody recovery. It is further shown that recovered antibodies are functional and compatible with downstream applications.

Literature Cited

Nidhi, N. et al. (2015) On-bead antibody-small molecule conjugation using high-capacity magnetic bead J. Immunol. Methods  http://dx.doi.org/10.1016/j.jim.2015.08.008

Cell-Free Expression Application: Antibody Screening

Posted on by Gary Kobs

TestPermissions2Ricin, derived from caster seeds, inhibits protein synthesis by binding to ribosomes, resulting in cell death. The protein is composed of two polypeptide chains: Ricin Toxin A chain and Ricin Toxin B chain. Ricin inhibits protein synthesis very quickly, and the cell or tissue damage begins within several hours. However, signs of poisoning often are not noted before significant damage has been done, making treatment difficult. Therapeutics that either block the ribosome binding site or compete with the toxin for binding are highly desired. Both antibodies and competitive ligands inhibited binding of the toxin to cell membranes.

A recent publication by Dong et al. (1), described a study to investigate the therapeutic effect of mAb 4C13, a monoclonal antibody against ricin. One of first experiments performed was to determine the general effect the inhibition of protein synthesis induced by ricin using cell-free expression.

In the study, the authors used T3 Coupled Reticulocyte Lysate Systems from Promega. Both ricin and mAb were diluted with saline. Aliquots of ricin (80 ng/ml) were mixed with an equal volume mAbs (1.6μg/ml) or saline alone and incubated at 4 °C for 1.5 h. A total volume of 4μl of sample was added into the reaction system (i.e, T3 Coupled Reticulocyte and plasmid DNA containing the lucifersase gene downstream of T3 RNA phage promoter). After incubation at 30 °C for 1.5 h, the products were cooled at −20 °C for 10 min. A total of 5μl of each reactive product containing synthesized luciferase was mixed with 50μl luciferase assay reagent pre-equilibrated to room temperature, and the fluorescence absorbance was measured immediately with the micro-ELISA Reader.

Positive results obtained from this preliminary experiment, led to more thorough experiments to determine the dosage effect using in vivo models (i.e., cell lines and mice) to characterize the cytotoxicity and binding activity of mAb 4C13. The mAB 4C13 was shown to be a effective in the mouse model.

Dong, N. et al. (2015) Monoclonal antibody , mAb 4C13, an effective detoxicant antibody against ricin poisoning. Vaccine 33, 3836–42

Sample preparation: A critical step for consistent protein phosphorylation data

Posted on by Gary Kobs

MSextractcroppedProtein phosphorylation is a very important protein post-translational modification that controls many cellular processes including metabolism, transcriptional and translation regulation, degradation of proteins, cellular signaling and communication, proliferation, differentiation, and cell survival (1). Approximately 35% of human proteins are phosphorylated. Phosphoproteins are low in abundance, and, therefore, are challenging to detect and characterize by mass spectrometry. Different enrichment systems have been developed to isolate phosphopeptides. Among these techniques, immobilized metal affinity chromatography (IMAC) using Fe3+ and Ga3+ has been widely used for the enrichment of phosphopeptides.

Typical experimental workflows are tedious and consist of numerous steps, including sample collection and cell lysis. One of the major challenges of the process is to maintain the in vivo phosphorylation state of the proteins throughout the preparation process

To evaluate the effect of sample collection protocols on the global phosphorylation status of the cell, a recent paper by Kashin et al. compared different sample workflows by metabolic labeling and quantitative mass spectrometry on Saccharomyces cerevisiae cell cultures (2).

Three different sample collection workflows were evaluated: two that used denaturating conditions and involved mixing of cell cultures with an excess of either ethanol (EtOH) at −80 °C or trichloroacetic acid (TCA), and a third under nondenaturing conditions and washing the cells in PBS.

Their data suggest that either TCA or EtOH sample collection protocols introduced lower collection bias than the PBS protocol. It was also suggested that similar studies be carried out to determine what effects sample preparation has on other post translation modifications such as acetylation or ubiquitination.

Literature Cited

  1. Thingholm T.E. et al, (2009) Analytical strategies for phosphoproteomics. Proteomics 9,1451–68
  2. Kanshin, E. et al. (2015)  Sample Collection Method Bias Effects in Quantitative Phosphoproteomics. J  Proteome Res. 14, 2998-04.

pH Reactive Dyes for Screening Antibody Internalization

Posted on by Gary Kobs
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Antibody drug conjugates (ADCs) are a new class of therapeutic drugs that uses antibodies to deliver highly toxic drug molecules specifically to the cancer cells. A key requirement for ADCs is the ability of antibody to bind to the cancer cells followed by internalization and subsequent release of drug inside the cells leading to cell apoptosis.

Traditionally, selection of lead antibody candidates for ADCs was done in a sequential workflow where antibodies were first selected based on their affinity followed by characterization involving antibody internalization and drug conjugation. However, there is evidence that high affinity doesn’t always correlate with good internalization and hence there is a need to screen antibodies for internalization properties in addition to their affinities.

Promega has developed a method that allows antibody to be screened for their internalization properties in a simple, plate-based format. The method uses pH sensor dyes (pHAb dyes), which are not fluorescent at neutral pH but become highly fluorescent at acidic pH. When antibody conjugated with pHAb dye binds to its antigen on the cancer cell membrane they are not fluorescent but upon internalization and trafficking into endosomal and lysosomal vesicles the pH drops and dye becomes fluorescent.

Fluorescence signal, for pHAb dyes conjugated using either amine or thiol chemistry, is minimal at pH>7 and increase significantly as the pH drops to pH 5.0, which is a typical pH in cell endosomal compartment. Moreover, pH response of free pHAb dye is similar to that of conjugated dye indicating that conjugation chemistry doesn’t influence the pH response of the dye.

Due to the high signal-to-background ratios of the dyes, plate-based internalization assays can be performed, enabling screening of large libraries of antibodies for their internalization properties, hopefully leading to improved identification of lead candidates for ADC applications.

Filter-Aided Sample Preparation before Mass Spec Analysis: An Evaluation of FASP and eFASP

Posted on by Gary Kobs

12271ma_800pxFilter-aided sample preparation (FASP) method is used for the on-filter digestion of proteins prior to mass-spectrometry-based analyses (1,2). FASP was designed for the removal of detergents, and chaotropes that were used for sample preparation. In addition, FASP removes components such as salts, nucleic acids and lipids. Akylation of reduced cysteine residues is also carried out on filter, after which protein is proteolyzed by use of trypsin on filter in the optimal buffer of the enzyme. Subsequent elution and desalting of the peptide-rich solution then provides a sample ready for LC–MS/MS analysis.

Erde et al. (3) described an enhanced FASP (eFASP) workflow that included 0.2% DCA in the exchange, alkylation, and digestion buffers,thus enhancing trypsin proteolysis, resulting in increases cytosolic and membrane protein representation. DCA has been reported (4) to improve the efficiency of the denaturation, solubilization, and tryptic digestion of proteins, particularly proteolytically resistant myoglobin and integral membrane proteins, thereby enhancing the efficiency of their identification with regard to the number of identified proteins and unique peptides.

In a recent publication (5) traditional FASP and eFASP were re-evaluated by ultra-high-performance liquid chromatography coupled to a quadrupole mass filter Orbitrap analyzer (Q Exactive). The results indicate that at the protein level, both methods extracted essentially the same number of hydrophobic transmembrane containing proteins as well as proteins associated with the cytoplasm or the cytoplasmic and outer membranes.

The LC–MS/MS results indicate that FASP and eFASP showed no significant differences at the protein level. However, because of the slight differences in selectivity at the physicochemical level of peptides, these methods can be seen to be somewhat complementary for analyses of complex peptide mixtures.

  1. Manza, L. L. et al. (2005) Sample preparation and digestion for proteomic analyses using spin filters Proteomics  5, 1742–74.
  2. Wiśniewski, J. R. et al. (2009) Universal sample preparation method for proteome analysis Nat. Methods 6, 359–62.
  3. Erde, J. et al. (2014) Enhanced FASP (eFASP) to increase proteomic coverage and sample recovery for quantitative proteome experiments. J. Proteome Res. 13, 1885–95.
  4. Lin, Y. et al. (2008) Sodium-deoxycholate-assisted tryptic digestion and identification of proteolytically resistant proteins Anal. Biochem.  377, 259–66.
  5. Nel. A. et al. (2015) Comparative Reevaluation of FASP and Enhanced FASP methods by LC-MS/MS/ J Proteome Res. 14, 1637–42.

Targeting MYC: The Need to Study Protein:Protein Interactions in Cells

Posted on by Michele Arduengo
Crystal Structure of MYC MAX Heterodimer bound to DNA ImageSource=RCSB PDB; StructureID=1nkp; DOI=http://dx.doi.org/10.2210/pdb1nkp/pdb;
Crystal Structure of MYC MAX Heterodimer bound to DNA ImageSource=RCSB PDB; StructureID=1nkp; DOI=http://dx.doi.org/10.2210/pdb1nkp/pdb;

In 1982, picked up because of its homology to chicken virus genes that could transform cells, MYC became one of the first human genes identified that could drive cellular transformation (1,2). Since that time countless laboratories have prodded and poked the human MYC gene, the MYC protein, their homologs in other animal models, and their transforming viral counterparts.

MYC is a transcription factor and forms heterodimers with a required protein partner, MAX, before binding to the E box sequences of DNA regulatory regions (3). MYC regulates gene expression of many targets through interactions with a host of proteins, often referred to as the MYC Interactome (2).  In fact, MYC is estimated to bind 10–15% of the genome, and it regulates the expression of genes that  are transcribed by by each of the three RNA polymerases (2).

MYC plays a central role in regulating cell growth, proliferation, apoptosis, differentiation and transformation, acting as a central integrator of cellular signals. MYC is tightly regulated at multiple levels from gene expression to protein stability. Dysregulation (usually upregulation) of the amount and stability of Myc protein is observed in many human cancers. Even in cancers in which MYC is not directly involved in transforming cells, its normal expression is often required to support the extracellular matrix and/or vascularization necessary for tumor growth and formation (4).

Because MYC is such a central player cancer pathology, it is an attractive target for cancer therapeutics  (2) .

Continue reading “Targeting MYC: The Need to Study Protein:Protein Interactions in Cells”

HaloTag® Research Application: Detection of Cancer Biomarkers

Posted on by Gary Kobs

10242TAAntibodies labelled with radioisotopes or the sequential administrationof an antibody and a radioactive secondary agent facilitate the in vivo detection and/or characterisation of cancers by positron emission tomography (PET) or by single-photon emission computed tomography (SPECT) imaging.

There are drawbacks to both methods, including prolonged exposure to radiation and  ensuring that both the antibody and the radiolabelled secondary agent are suitably designed so that they bind rapidly upon contact at the tumor.

A recent publication (1) investigated a alternative method utilizing the HaloTag® dehalogenase enzyme HaloTag® is a dehalogenase enzyme (33 kDa) that contains an engineered cavity designed to accommodate the reactive chloroalkane group of a HaloTag® ligand (HTL). Upon entering the enzyme cavity, the terminal chlorine atom rapidly undergoes nucleophilic displacement, and a covalent adduct is formed, effectively anchoring the HaloTag® ligand in a precise location.

Three new HaloTag® ligands were synthesized and each labelled with the SPECT radionuclide indium-111  111In-HTL-1  and the dual-modality HaloTag® ligands,111In-HTL-2 and111;In-HTL-3 containing TMR which allows complementary imaging data).

For the validation of the pretargeting strategy based on these HaloTag® ligands, the target human epidermal growth factor receptor 2 (HER2)was selected. Trastuzumab (Herceptin®) was selected as the primary targeting agent and was modified with HaloTag® protein via the trans-cyclooctene/tetrazine ligation.

All three 111In-labelled HaloTa®g ligands exhibited significantly higher binding to the HER2 expressing when compared to negative controls.

Literature Cited

Knight, J. C et al.(2015) Development of an enzymatic pretargeting strategy for dual-modality imaging. Chem. Commun. 51, 4055–8.

How Confident are You in Your Mass Spectrometry Data?

Posted on by Gary Kobs

Data generated by scientific instruments and decisions based on that data depend on optimal instrument performance. Clinical assays rely on mass spectrometric (MS) data for accurate results so that correct health related results are gained and appropriate results-based decisions are made. However, there are no generally agreed upon tools nor performance standards for mass spectrometry. Furthermore, while several software tools exist that serve to assist  with the analysis of instrument performance, a dedicated reagent software package has yet to be created. For optimal liquid chromatography (LC) performance, parameters like retention time, peak width and peak heightare typically reported. Commonly monitored MS parameters include mass accuracy, mass resolution, signal-to noise, sensitivity, limit of detection (LOD), limit of quantitation (LOQ) and dynamic range.

The 6 × 5 LC-MS/MS Peptide Reference Mix is designed for use in method development and optimization,and for routine liquid chromatography (LC) and mass spectrometry (MS) instrument performance monitoring. The product is a mixture of 30 peptides: 6 sets of 5 isotopologues of the same peptide sequence. The isotopologues (Figure 1) differ only by the number of stable, heavy-labeled amino acids incorporated into the sequence. The labeled amino acids consist of uniform 13C and 15N atoms. Each of the isotopologues is indistinguishable chemically and chromatographically. However, since they differ in mass, they are clearly resolved by mass spectrometry.

Figure 1.
Figure 1.

The isotopologues of each peptide are present in a series of tenfold differences in concentration or molar abundance. If 1pmol of the mixture is loaded onto an LC column, the next lighter isotopologue would be 100fmol, the next 10fmol, the second lightest 1fmol, and the lightest 100amol. This range allows assessment of the instrument’s dynamic range and sensitivity from a single run.

Peptides with a wide range of hydrophobicities were chosen to enable reporting of LC column performance. The most hydrophilic peptide gives users a tool to optimize the capture of hydrophilic peptides that might be difficult to capture otherwise, but that are too precious to use for method development.

To assist in data processing, a complementary software tool, is provided, the 6 × 5 LC-MS/MS Peptide Reference Mix Analysis Software (The PReMiS™ Software). The PReMiS™ Software produces a tabular report of calculated instrument parameters, graphical analysis of linearity curves as well as reporting the history of user-selected parameters such as LC retention time, peak height and mass accuracy. If the laboratory has a collection of instruments, there is also an option to compare parameters across instruments.

Improved Characterization and Quantification of Complex Cell Surface N-Glycans

Posted on by Gary Kobs
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N-Glycosylation is a common protein post-translational modification occurring on asparagine residues of the consensus sequence asparagine-X-serine/threonine, where X may be any amino acid except proline. Protein N-glycosylation takes place in the endoplasmic reticulum (ER) as well as in the Golgi apparatus.

Approximately half of all proteins typically expressed in a cell undergo this modification, which entails the covalent addition of sugar moieties to specific amino acids. There are many potential functions of glycosylation. For instance, physical properties include: folding, trafficking, packing, stabilization and protease protection. N-glycans present at the cell surface are directly involved in cell−cell or cell−protein interactions that trigger various biological responses.

The standard method used to profile the N-glycosylation pattern of cells is glycoprotein isolation followed by denaturation and/or tryptic digestion of the glycoproteins and an enzymatic release of the N-glycans using PNGase F followed by analysis mass spec. This method has been reported to yield high levels of high-mannose N-glycans that stem from both membrane proteins as well as proteins from the ER.(1,2)

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For those researchers interested in characterizing only cell surface glycans (i.e.,  complex N-glycans)  a recent reference has developed a model system using HEK-292 cells that demonstrates a reproducible, sensitive, and fast method to profile surface N-glycosylation from living cells (3). The method involves standard centrifugation followed by enzymatic release of cell surface N-glycans. When compared to the standard methods the detection and quantification of complex-type N-glycans by increased their relative amount from 14 to 85%.

  1. North, S. J. et al. (2012) Glycomic analysis of human mast cells, eosinophils and basophils. Glycobiology. 2012, 22, 12–22.
  2. Reinke, S. O. et al. (2011) Analysis of cell surface N-glycosylation of the human embryonic
    kidney 293T cell line. J. Carbohydr. Chem.  30, 218–232.
  3. Hamouda, H. et al. (2014) Rapid Analysis of Cell Surface N‑Glycosylation from Living Cells Using Mass Spectrometry. J of Proteome Res. 13, 6144–51.

Optimizing Tryptic Digestions for Phosphoproteomics Analysis

Posted on by Gary Kobs

11296971-DC-CR-KinaseProtein phosphorylation is the most widespread type of post-translational modification. It affects every basic cellular process, including metabolism, growth, division, differentiation, motility, organelle trafficking, membrane transport, muscle contraction, immunity, learning and memory (1,2). Protein kinases catalyse the transfer of the phosphate from ATP to specific amino acids in proteins. In eukaryotes, these are usually Ser, Thr and Tyr residues. Due to the development of specific phosphopeptide enrichment techniques and highly sensitive MS instruments, phosphoproteomics has enabled researchers to gain a comprehensive view on the dynamics of protein phosphorylation and phosphorylation based signaling networks.

Due to its high cleavage specificity, trypsin is the commonly used proteolytic enzyme in MS-based proteomics, cleaving peptides carboxyterminal of the amino acids lysine and arginine. However, various factors such as the tertiary structure of a protein, adjacent basic amino acids or negatively charged residues close to cleavage sites as well as PTMs are known to impair proteolysis.

To gain closer insights into the impact of phosphorylation on tryptic digestion, a recent publication(3) systematically characterized the digestion efficiency of model peptide sequences that are known to be prone to incomplete digestion.

The results indicated that increasing trypsin concentrations up to a trypsin to peptide ratio of 1:10 led to a significant gain (1) in the overall number of phosphorylation sites (up to 9%) and in the intensities of individual phosphopeptides, thereby improving the sensitivity of phosphopeptide quantification.

The effect of organic solvents (ACN, acetonitrile and TFE trifuorethanol was also evaluated). Positive results were noted with TFE when determining the digestion of individual peptides. However TFE interfered with TiO2 phosphopeptide enrichment and therefore was not recommended for use with complex samples.

  1. Engholm-Keller, K and Larsen, M.R. (2013) Technologies and challenges in large scale phosphoroproteomics. Proteomics 13, 910–31.
  2. Beausoleil, S. A. et al. (2010) Tissue-specific atlas of mouse protein phosphorylation and expression. Cell 143, 1174–89.
  3. Dickhut, C. et al. (2014) Impact of Digestion Conditions on phosphoproteomics. J. Proteome Res. 13, 2761–70.