Post Translational Modification
Most proteins undergo some form of modification following translation. These modifications result in mass changes that are detected during analysis (Chart 1). Post-translational modifications such as glycosylation, phosphorylation, and sulfation, to name a few, serve many functions. As a result, the analysis of proteins and their post-translational modifications is particularly important for the study of diseases where multiple genes are known to be involved, such as heart disease, cancer and diabetes.
Phosphorylation
Reversible phosphorylation is one of the most important and well-studied post-translational modifications. Most commonly occurring on threonine, serine and tyrosine residues, phosphorylation plays critical roles in the regulation of many cellular processes including: cell cycle, growth, apoptosis and differentiation. Thus, the identification and characterization of phosphorylation sites is crucial for the understanding of various signaling events. Mass spectrometry (MS) of phosphopeptides obtained from tryptic protein digests has become a powerful tool for characterization (Stensballe, A., et al., 2000). However, there is a general need to significantly enrich samples for phosphopeptide content in order to compensate for low abundance, poor ionization, and suppression effects (Zhou, W., et al., 2000).
Immobilized metal affinity chromatography (IMAC) has been commonly used for purification of phosphorylated compounds. Our PHOS-Select™ Iron Affinity Gel is prepared with a novel iron [Fe(III)] chelate matrix based on our proprietary (patent pending) NTA analog chelate ligand. That matrix provides high capacity affinity binding of molecules containing phosphate groups, making these products ideal for the enrichment of phosphopeptides from protein tryptic digests, or small organic phosphocompounds (e.g. adenosine 5’-monophosphate). They can also be used for direct transfer of phosphocompounds for analysis by HPLC or mass spectrometry.
Glycosylation
One of the most common post-translational modifications of proteins is glycosylation, the covalent attachment of oligosaccharides. Carbohydrates in the form of asparagine-linked (N-linked) or serine/threonine-linked (O-linked) oligosaccharides are major structural components of many cell surface and secreted proteins. Glycoproteins play crucial roles in cellular processes such as protein sorting, immune recognition, receptor binding, inflammation, and pathogenicity.
The diversity of oligosaccharide structures often results in heterogeneity in the mass and charge of glycoproteins. This complex nature of glycosylation presents problems for proteomic analysis. We simplify the analysis of glycoproteins by offering deglycosylation kits, a number of individual glycosidases and several glycosylation inhibitors. Glycoprotein stains, labeled lectins and lectin resins are also available for the detection or purification of glycoproteins.
Enrichment of Biotinylated Peptides in Proteomics Application (e.g. ICAT)
Our Streptavidin HC (High Capacity) multiwell plates are prepared with a highly-porous, high-density streptavidin coating. Streptavidin has similar biotin-binding characteristics as avidin. Streptavidin can bind 4 moles of biotin per mole of protein with high selectivity and affinity (Kd~10-15). Unlike avidin, streptavidin has a near neutral pI that alleviates non-specific binding commonly associated with the basic avidin protein.
Chart of mass changes resulting from typical post-translational modifications of proteins and peptides. | ||
|---|---|---|
| Modification | Monoisotopic Mass Change | Average Mass Change |
| Homeserine formed from Met by CNBR treatment | -29.99281 | -30.0935 |
| Pyroglutamic acid formed from Gln | -17.02655 | -17.0306 |
| Disulphide bond formation | -2.01565 | -2.0159 |
| C-terminal amide formed from Gly | -0.98402 | -0.9847 |
| Deamidation of Asn and Gln | 0.98402 | -0.9847 |
| Methylation | 14.01565 | 14.0269 |
| Hydroxylation | 15.99491 | 15.9994 |
| Oxidation of Met | 15.99491 | 15.9994 |
| Formylation | 27.99491 | 28.0104 |
| Acetylation | 42.01056 | 42.0373 |
| Carboxylation of Asp and Glu | 43.98983 | 44.0098 |
| Carboxyamidomethylcysteine (Cam) from Cys (iodoacetamide) | 57.02146 | 57.0520 |
| Carboxymethylcysteine (Cme) from Cys (iodoacetic acid) | 58.00548 | 58.0367 |
| Phosphorylation | 79.96633 | 79.9799 |
| Sulphation | 79.95682 | 80.0624 |
| Pyridylethyleysteine (PE-Cys) from Cys (4-vinylpyridine) | 105.05785 | 105.1393 |
| Cysteinylation | 119.00410 | 119.1442 |
| Pentoses (Ara, Rib, Xyl) | 132.04226 | 132.1161 |
| Deoxyhexoses (Fuc, Rha) | 146.05791 | 146.1430 |
| Hexosamines (GalN, GlcN) | 191.06881 | 161.1577 |
| Hexoses (Fru, Gal, Glc, Man) | 162.05282 | 162.1424 |
| Lipoic acid (amide bond to lysine) | 188.03296 | 188.3147 |
| N-acetylhexosamines (GalNAc, GlcNAc) | 203.07937 | 203.1950 |
| Farnesylation | 204.18780 | 204.3556 |
| Myristoylation | 210.19836 | 210.3598 |
| Biotinylation (amide bond to lysine) | 226.07760 | 226.2994 |
| Pyridoxal phosphate (Schiff Base formed to lysine) | 231.02966 | 231.1449 |
| Palmitoylation | 238.22966 | 238.4136 |
| Stearoylation | 266.26096 | 266.4674 |
| Geranylgeranylation | 272.25040 | 272.4741 |
| N-acetylneuraminic acid (Sialic acid, NeuAc, NANA, SA) | 291.09542 | 291.2579 |
| Gluthathionylation | 305.06816 | 305.3117 |
| N-glycolylneuraminic acid (NeuGc) | 307.09033 | 307.2573 |
| 5'-Adenosylation | 329.05252 | 329.2091 |
| 4'-Phosphopantetheine | 339.07797 | 339.3294 |
| ADP-ribosylation (from NAD) | 541.06111 | 541.3052 |
| Methyl (Me) | 14.01565 | 14.0269 |
| Formyl (CHO) | 27.99491 | 28.0104 |
| Ehtyl (Et) | 28.03130 | 28.0538 |
| Acetyl (Ac) | 42.01056 | 42.0373 |
| t-Butyl (t-Bu) | 56.06260 | 56.1075 |
| Anisyl | 90.04695 | 90.1246 |
| Benzyl (Bzl) | 90.04695 | 90.1246 |
| Trifluoroacetyl (Tfa) | 95.98230 | 96.0087 |
| N-hydroxysuccinimide (ONSu, Osu) | 97.01638 | 97.0734 |
| T-Butyloxycarbonyl (Boc) | 100.05243 | 100.1173 |
| Benzoyl (Bz) | 104.02621 | 104.1082 |
| 4-Methylbenzyl (Meb) | 104.06260 | 104.1515 |
| Thioanisyl | 106.02411 | 106.1912 |
| Benzyloxymethyl (Bom) | 120.05751 | 120.1509 |
| 4-Nitrophenyl (Onp) | 121.01638 | 121.0954 |
| Benzyloxycarbonyl (Z) | 134.03678 | 134.1344 |
| 2-Nitrobenzoyl (NBz) | 149.01129 | 149.1058 |
| 2-Nitrophenylsulphenyl (Nps) | 152.98845 | 153.1614 |
| 4-Toluenesulphonyl (Tosyl, Tos) | 154.00885 | 154.1894 |
| Pentafluorophenyl (Pfp) | 165.98419 | 166.0501 |
| Diphenylmethyl (Dpm) | 166.07825 | 166.2224 |
| 2-Chlorobenzyloxycarbonyl (Cl-Z) | 167.99781 | 168.5792 |
| 2, 4, 5-Trichlorophenyl | 177.91438 | 179.4320 |
| 2-Bromobenzyloxycarbonyl (Br-Z) | 211.94729 | 213.0305 |
| 9-Fluorenylmethyloxycarbonyl (Fmoc) | 222.06808 | 222.2432 |
| Triphenylmethyl (Triyl, Trt) | 242.10955 | 242.3202 |
| 2, 2, 5, 7, 8-pentamehylchroman-6-sulphonyl (Pmc) | 266.09767 | 266.3611 |
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