Protein Structure Group
Section leader
Ulf Hellman
Ulf.Hellman(AT)LICR.uu.se
Phone +46 18 160423
Our main mission is to support the activities within the Branch with our specialized techniques, and when possible, to interact with other Branches in Sweden and abroad. The Protein Structure Group has solid experiences in peptide synthesis, radio-labeled amino acid sequencing and mass spectrometry. For the latter activity, we use a top of the line matrix-assisted-laser-desorption-ionization-time-of-flight-mass-spectrometer (MALDI-TOF-MS). The present instrument (Bruker Ultraflex III TOF/TOF), installed in March 2007, offers a striking sensitivity and accuracy for MS and especially for MS/MS, user friendliness and peptide sequencing possibilities. This latter property is a consequence of the extra "TOF" which allows development of full fragment spectra in few seconds. Our three main activities are described in more detail below.
Peptide synthesis
Contrary to the trend of outsourcing this highly specialized skill, we prefer to take advantage of our long gained experience. Our synthesizer, a ten-year-old Applied Biosystems 433A instrument, is operated with Fmoc chemistry, and produces high quality peptides. The synthesis of peptides modified with phosphorylations, acetylations, oxidations or other chemical groups at given amino acid residues, has been most useful for the different groups at our Branch. All peptides are worked up manually and often, depending on the intended use, purified to homogeneity by liquid chromatography. The products are quality controlled using MALDI-TOF/TOF-MS. The peptides produced are used to generate anti-peptide antibodies, as substrates or inhibitors, or as ligands in affinity chromatography experiments. An important extension has developed over the last years – we now also carry out affinity purification of anti-peptide antibodies in collaboration with other groups of our Branch. Another, now well established development in our Group is that we, in collaboration with other Groups, perform affinity based searches for interacting partners using immobilized ligands (i.e. a modified synthetic peptides). The combination of the biotin-streptavidin system and magnet beads has showed to be very powerful.
Radiolabeled phosphopeptide mapping
The introduction in our group of MALDI-TOF-MS twelve years ago has made peptide sequencing (see below) faster, more sensitive and at dramatically lowered running costs, compared to classical chemical amino acid sequence analysis ("Edman degradation"), a technique we no longer employ. However, the designated peptide sequencer has been remodeled to perform position analysis of phosphorylated (³²P) Tyr, Ser and Thr residues in known proteins.
MALDI mass spectrometry
Sample preparation for mass spectrometry
Over 95 % of the samples for analysis by MALDI-TOF/TOF-MS are delivered as bands or spots from one- or two-dimensional SDS-PAGE gels. The gel separated proteins are prepared for MS analysis by in-gel tryptic digestion. With Coomassie-visible material only a few percent of the digest is needed for analysis; with silver-stained material, often the entire sample must be applied after concentration and desalting on micro RPC columns (i.e. C18 µZipTip). We have recently introduced a novel approach to enrich for acidic, especially phosphorylated peptides, highly important but notorious for low sensitivity by MALDI TOF MS. This is based on home-made micro-columns comprising TiO2 particles. Recently we were offered to test a novel approach – pipette tips covered with TiO2/ZrO2 and this has proven to further increase the specificity for this group of highly relevant peptides.
Peptide mass fingerprinting (PMF) and analysis of Post Translational Modification (PTM)
Determining protein identity by PMF is a routine procedure for known proteins. After the generation of a proteolytic digest and determination of peptide masses by MALDI-TOF/TOF-MS, we employ a search engine (ProFound or MASCOT are preferred) to search a match with a protein in the sequence databases. If a significant mass spectrum is obtained, we are practically always able to identify the protein with high confidence. However, even when a significant match is found, we often confirm the identity by subjecting a few peptides to fragment analysis by Post Source Decay (see below) followed by MS/MS search via MASCOT. Should we fail to identify the target protein, the identity may be established by obtaining the amino acid sequence of one or more tryptic peptides (see below). A sequence homology search by BLAST is, in contrast to PMF, tolerant to amino acid substitutions. We have taken up a technique of Lys-modification (an imidazol derivative), which renders lysine containing peptides more basic and therefore increases the sensitivity resulting in higher sequence coverage. As a bonus, such peptides are easy to fragment for sequence analysis. Furthermore, the Lys-Tag reagent may be used to label a set of proteins with deuterium for relative quantifications. Over the last years, we have carried out interesting proteomics projects with groups at e.g. the Karolinska Hospital looking for proteins that are significantly changed in various tumors. We also spend a fair amount of effort to study PTMs, which are of crucial importance in signal transduction. The nature of the modification determines the difficulty involved – we have been successful with e.g. methylation, acetylation, ubiquitinylation and phosphorylation.
Post Source Decay (PSD) based peptide sequencing by MALDI-TOF/TOF-MS
Fragment analysis of peptides by MALDI-TOF/TOF-MS using PSD is a straightforward technique. The resulting fragment spectra are commonly used for protein identification, but are difficult to use for de novo sequencing. The TOF/TOF technology allows complete PSD spectra to be generated in seconds, so it is possible to scan through several peptides from one digest in a short time. This easy and quick PSD combines extremely well with the “Chemically Assisted Fragmentation” (CAF) derivatization protocol, leading to easily interpreted spectra, as they comprise a unique series of y-ions. Hence, an amino acid sequence can unambiguously (with the single exception of the isobaric Leu/Ile) be determined faster, cheaper and more sensitive than was ever possible using chemical Edman degradation. We use CAF-PSD for identification of un-characterized species, as well as for analysis of modified peptides. An example where we identify a phospho-Ser by sequencing after sulfonation is given in the figure. As the CAF reagent was discontinued early 2008, we have started to replace it by SPITC (4-sulfophenyl-isothiocyanate), which is a much cheaper compound with similar positive effects on peptide fragmentation by MALDI-TOF/TOF.
The peptide D F I/L D S I/L F S T R is phosphorylated on a Ser residue, but as it has two Ser, we cannot, based on the added 80 Da, tell which Ser is modified. By PSD sequencing after sulfonation, it was clearly shown that the second Ser carries the modification. The same sequence appears in two versions – the upper one starts by the loss of 136 from the sulfonation leaving the pSer intact; the lower is initiated by a “neutral loss” of 98 (H3PO4)from the pSer leaving 69 Da for that residue. (Taken from Fraczyk et al. Bioorg Chem.;38,124-131 (2010).
| Group Members: | |
| Position | Name |
| Senior Technical Assistant | Ulla Engström |
| Selected Publications (see also annual reports): | ||
| Conrotto, P., and Hellman, U. Lys Tag: an easy and robust chemical modification for improved de novo sequencing with a matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometer. Rapid Commun Mass Spectrom 22, 1823-1833 (2008) |
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| Hellman K, Alaiya AA, Becker S, Lomnytska M, Schedvins K, Steinberg W, Hellström AC, Andersson S, Hellman U, Auer G. Differential tissue-specific protein markers of vaginal carcinoma. Br J Cancer. 100, 1303-14 (2009). |
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| Nagy N, Matskova L, Kis LL, Hellman U, Klein G, Klein E. The proaptotic function of SAP provides a clue to the clinical picture of X-linked lymphoproliferative disease. Proc Natl Acad Sci U S A. 106, 11966-71 (2009( |
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| Rahman-Roblick R., Hellman, U., Becker S., Bader, FG., Auer, G., Wiman, K., and Roblick, UJ. Proteomic identification of p53-dependent protein phosphorylation. Oncogene 27, 4854-4859 (2008) |
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| Larsson A, Söderberg L, Westermark GT, Sletten K, Engström U, Tjernberg LO, Näslund J, Westermark P. Unwinding fibril formation of medin, the peptide of the most common form of human amyloid. Biochem Biophys Res Commun. ;361, 822-8.(2007) |
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| Lennartsson J, Wardega P, Engström U, Hellman U, Heldin CH. Alix facilitates the interaction between c-Cbl and platelet-derived growth factor beta-receptor and thereby modulates receptor down-regulation. J Biol Chem. 281, 39152-8 (2006). |
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| Bergström J, Engström U, Yamashita T, Ando Y, Westermark P. Surface exposed epitopes and structural heterogeneity of in vivo formed transthyretin amyloid fibrils. Biochem Biophys Res Commun. 348,:532-9. (2006) |
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| Persson C, Kappert K, Engström U, Ostman A, Sjöblom T. An antibody-based method for monitoring in vivo oxidation of protein tyrosine phosphatases. Methods. 35, 37-43 (2005) |