Cambridge University
A green valley surrounded by snow-topped mountains, a blue sky, and golden sunshine was the setting for the ABRF workshop held in Davos, Switzerland in conjunction with the First European Protein Society Meeting on May 28, 1995. The temptation to go hill-walking was resisted by at least 120 people, and the start was hampered by a cycle race through Davos, which delayed some delegates, but by the second talk the hall was stocked with eager listeners.
This workshop was organized by Peter Hunziker and Len Packman. Opening introductions were given by Greg Grant and Len Packman and then the meeting began with presentations in five separate subject sections. Chairpersons during the meeting were Peter Hunziker, Greg Grant, Len Packman, and Richard Simpson.
Chromatography
Richard Simpson, Ludwig Institute, Melbourne, Australia, "Capillary HPLC: A Tool for Protein Structure Analysis".
Capillary HPLC seems to be underused, but Richard described how easily conventional HPLC apparatus can be adapted to perform well and inexpensively. A flow splitter is used between the pumps and the injector to produce the desired flow rate, and a U-shaped piece of capillary tubing (with the polyamide layer burned away) forms the basis of the flow cell. ("Kits" can be purchased from LC Packings.) Columns can be bought commercially but can also be easily made in the laboratory for a fraction of the cost using a Shandon column packer, capillary tubing, Swagelok end fittings, and a 50 x 2.1 mm stainless steel reservoir. A slurry of medium in propanol is packed for 30 min at 300 bar, and conditioned in 50% aqueous methanol for 30 min; the column is then ready to use.
Richard has also studied the effect of column length and showed that a 10 mm long capillary can be just as effective as a 250 mm one. Examples were given of the use of conventional silica-based supports (e.g., Brownlee RP-300) whose tolerance of fast flow rates at moderate pressures allows rapid separation of complex mixtures (e.g., peptide maps), reducing a 60 min separation to only 12 min without any compromise in resolution. This capillary chromatography set-up is readily interfaced with mass spectrometry for analyzing digests of protein spots from gels or electroblots.
Christopher Southan, SmithKline Beecham, Welwyn, UK, "Fast HPLC on Cheap, Disposable Columns".
Chris described a different method for producing capillary HPLC columns in the laboratory. Using TEFZEL or PEEK tubing and 1/16" Kel-F ferrules, frits, and end-fittings that are available from Upchurch or Alltech, microbore columns with 0.5 to 2.5 mm internal diameters can be made at very low cost. The columns can be dry- or slurry-packed with conventional silica, macroporous, ion-exchange, or size-exclusion media. Columns are dry-packed by filling the tubing with media to about two times the desired final length, pumping solvent through the tubing until the media has settled, and then cutting the tubing to the desired length-no inlet frit is needed. Slurry packing is done with a Poros self-pack reservoir.
An advantage of macroporous media (e.g., Poros, PLRPS, Resource, etc.) is rapid cycle time between sample injections. Gradient HPLC screening assays can be as short as 5 min at 0.5 ml/min, and size-exclusion columns can be used for quick sample desalting. With Poros-based columns, proteins irreversibly bound to reverse phase supports can be applied directly to protein sequencers after extruding media from columns and can be mass analyzed by MALDI/TOF after mixing with matrix.
Quality Control
Len Packman, Cambridge University, UK, "The Detection and Prevention of Aspartimide Formation in Solid Phase Peptide Synthesis".
Len described work directed at solving the problem of aspartimide formation in peptide synthesis. This acid- or base-catalyzed reaction can occur when the amide nitrogen carboxyl-terminal to Asp attacks the [[beta]]-carboxylate group of Asp to form a succinimide ring. The succinimide ring can then be opened by reaction with piperidine or water, giving predominantly the [[beta]]-peptide, but also some [[alpha]]-peptide and racemization of Asp. Once aspartimide rings have formed, there is no simple way to recover the desired peptide at high yield.
The only effective way to detect aspartimide formation is through mass analysis. The succinimide ring results in an 18 Da loss in mass, and the piperidine adduct a 67 Da gain. Amino acid analysis will not detect either of these. Several methods have been proposed to reduce the incidence of aspartimide formation, but they have only suppressed, not eliminated, this side reaction. In Fmoc-t-butyl chemistry, the most susceptible sequences are: Asp-Asn, Asp-Gly > Asp-Gln, Asp-Ala. This side reaction occurs at a low level at each cycle and is cumulative, so susceptible sequences close to the carboxyl-terminus will be more problematic than those near the amino-terminus.
Len has addressed this problem through the use of the reversible protecting group 2-hydroxy-4-methoxybenzoyl (Hmb) at the reactive amide nitrogen. This is introduced as the N,O-bisFmoc-N-(2-hydroxy-4-methoxybenzyl) amino acid OPfp ester and then aspartic acid is coupled as a symmetric anhydride, followed by normal synthesis (any activation chemistry) after the susceptible sequence. The approach effectively eliminates aspartimide formation, and the Hmb group is removed during normal deprotection and cleavage with trifluoroacetic acid. Another advantage is that the Hmb group suppresses [[beta]]-sheet formation, acting as a disaggregant and enhancing the synthesis of difficult sequences. The O-acetylated form of Hmb is not removed by trifluoroacetic acid but acts as a solubilizing agent, allowing the purification of previously insoluble peptides. Ac-Hmb can then be removed from the purified peptide by O-deacetylation with piperidine and cleavage with trifluoroacetic acid.
Finn Kirpekar, Odense University, Denmark, "MALDI Mass Spectrometry of Nucleic Acids".
Finn described the immense progress that has been made in analysis of oligonucleotides by MALDI mass spectrometry. One of the more significant developments has been the use of 3-hydroxypicolinic acid as a matrix with machines equipped with ultraviolet lasers, but certain precautions must be taken to ensure good results. As polyanions, DNA and RNA strongly bind Na+ and K+, and these must be removed before analysis. Samples can be treated with ion-exchange resin beads to exchange Na+ and K+ for NH4+. During ionization, ammonia dissociates to give the molecular ion.
In positive ion mode the charge is on the base, and in negative ion mode it is on the phosphate group. A resolution of 810 has been achieved at 5-6 kDa using a reflector. However, use of a reflector can produce fragmentation ions, so linear analysis is preferred for samples more than 50 bases long. Fragmentation seems to occur through base loss followed by cleavage of the phosphodiester backbone at the 3' C-O bond. Oligo-dT (up to a 70-mer) is very stable during reflector analysis because base loss is initiated by protonation, which cannot occur with T, and RNA is more stable during analysis than DNA. These approaches can be used to sequence oligonucleotides that have been treated briefly with 5'- or 3'-exonucleases, to generate sequence ladders during MALDI analysis.
Post-Translational Modification
Alastair Aitken, National Institute for Medical Research, Mill Hill, UK, "Identification of Post-Translational Modifications; Synthesis of Modified Peptides and Production of Specific Antisera".
Alastair described work aimed at identifying the post-translational modifications of 14-3-3 proteins, which interact with a large number of kinases and other proteins and are present in many isoforms. There are fewer gene products than previously thought, because two sets of isoforms have the same sequence but differ by post-translational modification.
To identify the modification, the proteins were analyzed by ESMS, but they gave weak signals. This was overcome by developing an in-line trapping column to bind dilute protein samples and wash away salts and other interfering compounds. When the column is switched in-line, the ESMS solvent (e.g., acidified 50% acetonitrile or gradients of acetonitrile to separate peptide/protein mixtures) elutes samples as sharp peaks that give high quality signals. A Poros 20 um support in a 0.25 mm capillary column gives a good signal with less than 500 fmol of protein. Using this approach, the a and b forms of the protein were shown to differ by 80 Da, and the suspected phosphorylation site was identified by digestion and LCMS analysis using 10-80% acetonitrile gradients to separate digest mixtures.
Other traps that have worked well include a column from Michrom for removing SDS from samples eluted from gels. For those interested in X-ray analysis, a method for treating crystals with the cold vapor from liquid N2 was described that allows a lifetime in the X-ray beam of several hours rather than just a few minutes.
Jan van Oostrum, Ciba, Basel, Switzerland, "Analysis and Profiling of Oligosaccharides of Immunoglobulin G1 Antibodies Using Electrospray LC/MS and High pH Anion Exchange Chromatography".
When recombinant IgG antibodies are produced commercial-ly by a batch process, they are expressed in cells that have a different glycosylation machinery from human cells, so it is important to be able to characterize the post-translational modifications of these proteins precisely and quickly. Jan described a system that allows variation in glycosylation patterns to be elucidated within a single day, so the results can be relayed to the manufacturing process and the growth conditions adjusted to ensure a homogeneous product.
The IgG is digested with trypsin, and the fragments are separated and analyzed by LCMS. However, the ESMS is configured so that as the quadrupoles scan the mass range, there is a stepped change in the orifice voltage; the lower mass range (m/z 100-400) experiences orifice voltage fragmentation, with formation of carbohydrate-specific fragments, while the rest of the mass range (m/z 450-2400) does not. Two sets of data are therefore collected from each scan, and specific sugars can be detected by their unique signature from the fragmentation part of the scan, e.g., an m/z of 204 indicates a N-acetylglucosamine residue. Both the location and the structure of the glycosylation sites are readily deduced.
Mass Spectrometry
Darryl Pappin, Imperial Cancer Research Fund, London, UK, "Chemical/MALDI Approaches to the Analysis of Peptides at the Sub-Picomole Level".
Darryl described a set of reagents he developed to facilitate ESMS and MALDI analysis of peptide structure and composition at low pmol to fmol levels. Trifluoroethyl isothiocyanate (TFEITC) is a volatile compound that allows manual Edman degradation of peptides. Instead of the solvent extraction steps that accompany the use of PITC, this reagent is removed by vacuum, and HFBA or TFA is used to cleave the derivatized residue.
This method can be adapted to generate sequence ladders for analysis by MALDI as follows. First decide how many cycles of sequence are needed, and remove an aliquot of the sample. Perform a cycle of Edman degradation on the aliquot using TFEITC to generate the n-1 peptide. Then add an equivalent aliquot to the first one, and repeat the Edman degradation to produce the n-1 and n-2 peptides. After repeating the process several times, the peptide mixture is analyzed by MALDI, and the difference in peak masses defines the sequence. Several residues of sequence can be obtained from 1 pmol of peptide.
Darryl also described some amino-terminal tagging reagents that enhance the detection of fragment ions during MALDI/post-source decay and during MS-MS. A quaternary ammonium alkyl-N-hydroxysuccinimide ester is used for post-source decay, but an analogous pyridine compound is needed for the low energy collisions in triple quadrupole MS-MS. In MALDI, post-source decay occurs through high and low energy collisions, and tagged material gives a cleaner fragmentation spectrum consisting mainly of a and d ions. However, these spectra are not as clear as the low energy MS-MS data, where complete series of b ions have been demonstrated for 13-residue peptides.
Both these reagents form part of an integrated approach to structure analysis. It is now possible to determine the mass of a peptide, then perform a cycle of Edman degradation with TFEITC to give the first residue and ascertain if any internal lysines are present, then esterify to determine the Asp and Glu content, then tag with the hydroxysuccinimide ester to do fragmentation studies, all starting with less than 1 pmol of material. All this information increases, by orders of magnitude, the fidelity of any database searches to identify a protein from its peptides.
Peter Roepstorff, Odense University, Denmark, "Full Characterization of the Covalent Structure of Proteins by Mass Spectrometry".
Peter presented work done in his laboratory aimed at the full characterization of the covalent structure of proteins by mass spectrometry. In validating the integrity of recombinant proteins, the most productive approach by far is proteolytic digestion and LCMS analysis of the products. This routinely allows the entire sequence to be checked quickly, usually in no more than a day or so for "familiar" proteins.
There are many examples of unexpected point mutations in expressed proteins. Some can be detected through a change in mass of the whole protein, but if the mass change is only a few Da, the digestion-LCMS route is the only reliable way of screening for errors. MALDI analysis of digests has proved useful in the detection of disulfide bridges in proteins, and sequence information can often be obtained by MALDI, not through a post-source decay approach but through a much older technique: short acid hydrolysis can generate a ladder sufficient for a few residues of sequence.
In addition a few unusual modifications have been found, and an example was given of a chitinase in which, according to cDNA sequencing, there are PTTPTTPTT sequences; the prolines were hydroxylated and O-glycosylated with xylose. Through MALDI approaches, the post-translational modifications and four of seven disulfide bridges were assigned. Also, MALDI analysis after treatment with specific glycosidases provided the structure of N-glycosylated peptides from an allergen.
Christoph Eckerskorn, Max-Planck Institute, Martinsried, Germany, "MALDI Mass Spectrometry of Proteins Electroblotted after Polyacrylamide Gel Electrophoresis".
Christoph discussed the issue of obtaining masses from proteins immobilized on electroblots. Several groups have explored this area but few have been able to obtain data of the quality presented here.
The main technical problem is surrounding efficiently separated proteins with matrix. To analyze blotted proteins by MALDI, a high-capacity, high-density membrane is needed to ensure that protein accumulates near the surface. Sadly for many of us with ultraviolet laser instruments, MALDI from blots works better with infrared lasers. Microscopic examination of a sample that has been analyzed with an infrared laser shows the laser tunnels into the membrane to leave a hole. The signal persists from PVDF, but it diminishes quickly when nitrocellulose is used. An important tip is not to dry the blot before the matrix is incorporated and not to stain it either; parallel tracks can be stained to locate the sample. Samples can be ionized after treatment with some stains, but broad protein signals are produced, shifted in mass by the addition of several dye molecules, which can be partially resolved for small proteins. With India ink or colloidal gold staining, the quality of the spectra is deteriorated, but the correct protein mass is obtained.
Chris also described a system for scanning bands in one- and two-dimensional gels. In one-dimensional gels, a single band may show a mass difference between the leading and trailing edges, showing that the gel system is capable of resolving very small changes in mass due to heterogeneity in glycosylation or other post-translational modifications or perhaps chemical modification by the gel system. With two-dimensional gel spots, mass analysis is performed in a two-dimensional grid, producing a contour map of the masses present. Mass signal intensity mirrored the density of stain on stained replicate gels. This system readily detected mass heterogeneity within spots but also detected overlapping spots that failed to stain. This process is suitable for automation and in the future one can envisage scanning entire two-dimensional gels for the masses of the proteins it contains.
Databases
Peter James, Federal Institute of Technology, Zurich, Switzerland, "Database Searches Using MS and MS/MS Data and Their Relevance to Core Facilities".
Peter provided an overview of databases available for protein identification. At a time when we are polarized into thinking about searching databases with protein fragments, Peter outlined the usefulness of using characteristics of intact samples purified by conventional means or by two-dimensional electrophoresis. A gel database on the ExPASy server on the World Wide Web can be searched by providing pI and mass or pI and gel position. Also, a search by amino acid composition alone can be productive if the data are of high quality. A technique of dual isotope labeling was described: organisms or cells are grown in the presence of specific 35S and 14C amino acids, and the 35S/14C ratios are measured after two-dimensional electrophoresis to obtain amino acid compositions for use in database searches.
Peter also reviewed the current status of "mass mapping" approaches, emphasizing how data can be improved to provide more accurate scores, through improved mass accuracy and the use of double digests. Deliberate chemical modifications are also helpful--such as methylation, acetylation, D2-exchange--because they give additional data on composition, even with peptide mixtures. The future, however, may lie in MS-MS data from LCMS with triple quadrupole and ion-trap detectors or from MALDI post source decay, either approach coupled to automated database searching without the need to interpret fragmentation spectra.
He concluded by providing addresses for databases and the sources of search software:
* MADMAE, Richard Johnson, MW and Edman data, software, E-mail: johnson@byron.u.washington.edu.
* PEPSEARCH, Darryl Pappin, mass fingerprint search, server, E-mail server: mowse@dl.ac.uk.
* MS-SEARCH, Peter James, mass fingerprint search, software/server, WWW at http://cbrg.inf.ethz.ch.
* PEPSEARCH, Matthias Mann, Tag/fingerprint search, enquire, E-mail: mann@embl-heidelberg.de.
* SEQUEST, John Yates, MS/MS search, enquire, E-mail: jyates@u.washington.edu.
* FRAGFIT, William Henzel, fingerprint search, software, E-mail: ckw@gene.com.
* GUESSPROT, search according to pI/MW, and AACOMPIT, Ron Appel, AAA search, server, WWW http://expasy.hcuge.ch/www/tools.html.
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