Protein Analysis and Purification

How Confident are You in Your Mass Spectrometry Data?

Posted on by Promega

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 Promega
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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 Promega

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.

Use of Cell-Free Technology to Evaluate Nuclease (TALEN) Activity on Target DNA

Posted on by Promega
ImageSource=RCSB PDB; StructureID=1qpf; DOI=http://dx.doi.org/10.2210/pdb1qpf/pdb;
ImageSource=RCSB PDB; StructureID=1qpf; DOI=http://dx.doi.org/10.2210/pdb1qpf/pdb;

Transcriptional activator-like effector nucleases (TALENs) have rapidly become a technique of choice for precision genome engineering. TALENs are custom-designed nucleases that consist of a modular DNA-binding domain fused to a monomeric, C-terminal FokI nuclease domain (1). TALENs work in pairs and are designed to recognize and bind to tandem-oriented sequences in genomic DNA, separated by a short spacer (15–30 bp). TALEN binding causes dimerization and activation of the FokI nuclease domains, which results in cleavage of the DNA within the spacer region. Small insertions or deletions (indels) are frequently introduced at this site, as the result of errors made during DNA repair by nonhomologous end-joining (NHEJ). These indels can be up to several hundred base pairs in length and result in frameshift mutations that lead to the production of truncated or nonfunctional proteins.

Successful use of TALENs for inducing targeted mutations has been reported in many conventional models, for example: mice, Xenopus and D. melanogaster. TALENs are also reported to be functional in a variety of other invertebrate arthropods, including mosquitos,silkworm and cricket. A recent publication (2) illustrates the use of TALEN technology for the genetic manipulation in P. dumerilii (marine ragworm).

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Discovering the Complexity of the Human Proteome

Posted on by Kelly Grooms

Transcription TranslationI should preface this blog by stating that I am a nucleic acids gal. My years in the lab were spent with tubes of DNA and RNA. In fact my one and only tentative foray into the field of protein resulted in a Western Blot so ugly that those who witnessed it have been sworn to secrecy. Given all of this, the mapping of the human proteome might seem like an odd topic for me to write about. Except that it isn’t really, because the sequencing of the proteome offers answers to some of the questions that the sequencing of the genome didn’t.

First, let’s start with what a proteome is: A proteome is all the proteins expressed at a certain time point. It can be as limited as the proteome of a single cell or as all encompassing as the proteome of an entire genome. However, unlike the genome, which is genetic information encoded in an organism’s DNA or RNA, the makeup of a proteome can vary dramatically as a result of expression patterns, alternative splicing events and post-translational modifications.

The genome is a constant, what you see today is what will still be there tomorrow. The proteome, on the other hand, is a constantly changing landscape. Up regulation or down regulation of a gene can mean more or less protein is present. Alternative splicing and post-translational modifications can result in fundamental changes to the protein itself.

In other words, if the genome is a beautiful, pristine Ansel Adams print, then the proteome is that same scene as interpreted by Andy Warhol—in Technicolor and 3D. Continue reading “Discovering the Complexity of the Human Proteome”

Purifying HIS-Tagged Proteins from Insect and Mammalian Cells

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MSextractcroppedMany different polypeptide fusion partners or affinity tags have been developed to facilitate purification of target proteins. The most commonly used tag for the purification and detection of recombinant expressed proteins is the His tag. Cloning vectors designed to generate His-tagged proteins contain 5–10 histidine residues at either the C- or N terminus of the expressed protein. The His tag adds only 0.84kDa to the mass of the protein and is nonimmunogenic. Also, because the tertiary structure of the tag is not important for purification, His-tagged proteins can be purified using native or denaturing conditions. The affinity of histidine residues for immobilized nickel allows selective purification of His-tagged proteins. The MagneHis™ Ni-Particles can bind up to 1mg of His-tagged protein per milliliter of particles providing a fast, efficient method for purifying His-tagged proteins with high yield and low background in a highly scalable format.

Bacterial expression of recombinant His-tagged proteins is a common technique. However, use of other systems, such as Sf9 insect cells,or HeLa or CHO mammalian cells for expression of recombinant proteins either intracellularly or secreted into the culture medium is increasing. These eukaryotic expression systems may allow more natural processing and modification of recombinant His-tagged proteins.
The following article:  illustrates the use of FastBreak™ Cell Lysis Reagent and the MagneHis™ Protein Purification System with insect and mammalian cell lysates. Proteins are purified from culture medium in the presence or absence of serum with only minior modifications to the standard protocol for bacterial cultures are required for purification from these diverse sources.
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Monitoring Mass Spec Instrument Performance and Sample Preparation

Posted on by Michele Arduengo, PhD

Proteomics, the analysis of the entire protein content of a living system, has become a vital part of life science research, and mass spectrometry (MS) is the method for analyzing proteins.  MS analysis of protein content allows researchers to identify proteins, sequence them and determine the nature of post translational modifications.

LC/MS performance monitoring. Each run used 1μg of human predigested protein extract injected into the instrument (Waters NanoAquity HPLC System interfaced to a Thermo Fisher Q Exactive™ Hybrid Quadrupole-Orbitrap Mass Spectrometer). Peptides were resolved with a 2-hour gradient. Weekly monitoring with the human extract ensured consistent analytical performance of the instrument.
LC/MS performance monitoring. Each run used 1μg of human predigested protein extract injected into the instrument (Waters NanoAquity HPLC System interfaced to a Thermo Fisher Q Exactive™ Hybrid Quadrupole-Orbitrap Mass Spectrometer). Peptides were resolved with a 2-hour gradient. Weekly monitoring with the human extract ensured consistent analytical performance of the instrument.

Mass spectrometry allows characterization of molecules by converting them to ions so that they can be manipulated in electrical and magnetic fields. Basically a small sample (analyte) is ionized, usually to cations by loss of an electron. After ionization, the charged particles (ions) are separated by mass and charge;  the separated particles are measured and data displayed as a mass spectrum. The mass spectrum is typically presented as a bar graph where each peak represents a single charged particle having a specific mass-to-charge (m/z) ratio. The height of the peak represents the relative abundance of the particle. The number and relative abundance of the ions reveal how different parts of the molecule relate to each other.

For the study of large, organic macromolecules, matrix associated laser desorption/ionization (MALDI) or tandem mass spec/collision induced dissociation (MS/MS) techniques are often used to generate the charged particles from the analyte. MS analysis brings sensitivity and specificity to proteome analysis. The technique has excellent resolution and is able to distinguish one ion from another, even when their m/z ratios are similar. Macromolecules are present in extremely different concentrations in the cells, and MS analysis can detect biomolecules across five logs of concentration.

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Shedding Light on Protein:Protein Interactions with NanoBRET™ Technique

Posted on by Michele Arduengo, PhD

NanoBRET™ TechnologyIf you are trying to investigate protein:protein interactions inside cells, you know how important physiologically relevant results are. If you overload your cells with fusion constructs, your protein interactions may not actually reflect what is going on in the cell, and if your BRET energy donor and acceptor do not have sufficiently separated spectra, you can pick up a fair amount of noise in your experiment. Using the new superbright NanoLuc® Luciferase, and the HaloTag® Technology, we have developed a sensitive BRET system to help you take a better look specific protein interactions that interest you. Promega research scientist, Danette Daniels, describes the system in the Chalk Talk below:

Optimize Your Western Blot

Posted on by Ben Schmidt
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Western Blot Detection.

You’ve probably been there. You’ve got a new antibody or you’re testing out one you’ve made yourself. After weeks or months of work, your antibody is going to help move your research project forward. As you excitedly head to the dark room to develop your film, your mood is crushed when you see…bands, more bands, and smears. Alas, science has played one more cruel joke on you as you experience what so many of your fellow scientists have before. Despite such a dismal beginning, you often can still get good western blots by changing steps in your protocol.

Several steps in the western blot protocol can be optimized.

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Mass Spec-Compatible Proteome Reference Material

Posted on by Promega
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The complexity of biological samples places high demand on mass spec analytical capability. Adequate monitoring of instrument performance for proteomics studies requires equally complex reference material such as whole-cell extracts. However, whole-cell extracts available commercially are developed for general research (e.g., enzymatic or Western blot analysis) and contain detergents and salts that interfere with reverse phase liquid chromatography and mass spectrometry. Even after clean up, the extracts need to be digested, requiring time, labor and experience to generate samples for use in mass spectrometry. To address the need for complex protein material, we have developed whole-cell protein extracts from yeast and human cells. The yeast extract offers the convenience of a relatively small and well annotated proteome, whereas the human extract provides a complex proteome with large dynamic range. The human extract also serves as reference material for studies targeting the human proteome.

The extracts are free of compounds that interfere with reverse phase liquid chromatography-mass spectrometry (LC-MS), and have been reduced with DTT and alkylated with iodoacetamide then digested with Trypsin/Lys-C Mix and lyophilized. These digested extracts (tryptic peptides) can be readily reconstituted in trifluoroacetic acid (TFA) or formic acid and injected into an instrument. The same human and yeast whole-cell extracts also are provided in an intact (undigested) form for users who would like to develop an independent method for preparing protein mass spectrometry samples. For convenience, the intact extracts are provided as a frozen solution.

Consistent extract protein composition is ensured by tight control over cell culture conditions and manufacturing process. Lot-to-lot consistency of extracts is monitored by various protein and peptide qualitative and quantitation methods, including LC-MS. (Quality control results are provided upon request.) Our manufacturing process assures compatibility with reverse phase liquid chromatography and mass spectrometry, minimal nonspecific protein fragmentation, nonbiological post-translational modifi cations and,for digested extracts, minimal undigested peptides. The extracts are optimized for a high number of peptide and protein identifications in mass spectrometry analysis.