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Sensitive peptide sequence analysis: a faster weigh-in for biomolecules
News
MOLECULAR
MEDICINE
TODAY,
JANUARY
1997
Sensitivepeptide sequenceanalysis: a faster weigh-in for biomolecules A new analytical technique developed by chemists in the USA should speed up the characterization and sequence analysis of minute quantities of peptides, proteins and other biomolecules. This has important implications for several branches of molecular medicine. Donald Hunt (Dept of Chemistry, University of Virginia, Charlottesville, VA, USA), a pioneer in the use of mass spectrometric methods for protein and peptide sequence analysis, has adapted a new liquid chromatography quadrupole (LCQ) ion-trap mass spectrometer from Finnigan MAT Corp. (San Jose, CA, USA) to make it even more sensitive. Hunt’s team was involved in the original conception of the LCQ, although Finmgan holds the patents. He and his team have now replaced tbe LCQ’s standard electrospray ionization source with a novel sheathlessnano-electmspray ionization source to increase its sensitivity. According to Hunt, the modified LCQ coupled with microcapillary high-performance liquid chromatography (HPLC) can be used to sequence as little as lo-‘s mol of a peptide. ‘The technique is 10 000 times more sensitivethan the classical microsequence analysis by Edman degradation,’ he says.This should allow a large number of problems in biochemistry, cell biology and immunology to be solved, because if a peptide sequence is known, the protein can often be identified from its genomic sequence. One such application involves identifying the peptide antigens recognized by cytotoxic T cells (CTLs; see Box 1). ‘This could be the tirst step in the preparation of vaccines that promote immunity against a variety of diseases,’ saysHunt. He and other collaborators (such as the groups of Victor Engelhard and Craig Slingluff, also at the University of Virginia) are using an approach that starts with eluting peptides from diseased cells. The peptides are then separated into fractions using HPLC, and these are assayedfor T-cell-stimulating activity. ‘By combining this T-cell assay with mass spectrometry,’ explains Hunt, ‘it is possible to define the mass of the peptide in the mixture that reconstitutes the epitope (the T-cell stimulatoty portion) recognized by the immune system.’ To tind the sequence of this peptide, the peptides in the biologically active fraction are separated, again using HPLC. The purified peptide is then injected directly into the ion-trap massspectrometer, where individual molecules are ionized and trapped if they fall within a set maximum and minimum mass-to-charge ratio. Trapped peptide ions are then bombarded with fast-moving helium
2
atoms, which randomly break them into smaller charged fragments at their amide bonds. The ion trap stores the fragments so that a much stronger signal than usual is produced in the mass spectrum. ‘This is where the power of this mass spectrometer lies,’ saysteam member Bob Settlage, ‘conventional MS devices look at only one ion at a time, while the ion trap stores all ions before scanning. From the spectrum of massesof these fragments, the researchers can work out the difference between two fragments differing by only a single amino acid. This means that they can defme the mass and thus the identity of the extra residue in the longer of the two fragments. When all the fragments have been analysed, the total sequence of amino acids in the original peptide can be deduced. ‘The ion trap also wins because it can fragment fragments,’ adds Settlage, ‘allowing the deduction of sequences that otherwise would be difficult to determine.’ The most challenging part of this approach is to analyse the minute quantities of immunostimulatory peptides present on the surfaces of diseased cells. These cells might be available in relatively small quantities and are often very expensive to grow up into larger quantities. CTLs are extremely sensitive: they can recognize one copy of a particular peptide per cell. This equates to only 1.5 X IO-t5 mol of peptide on lo9 cells. The microspray-HBLC-ion-trap technology has already been used to sequence lo-t6 mol of a tissue transplantation antigen. The reported results were obtained from 160 000 of a sample of 50 X 10’ cells. Although they haven’t yet repeated this procedure on a smaller number of cells, they expect to get the same results using 10’ cells. The availability of suitable CTLs and the numbers of CTLs required per assaycan also be a limiting factor in this approach, so any
Copyright
01997
economy resulting from increased sensitivity is very useful indeed. If the tiny amounts of peptide antigens produced as a result of viral infection, cancer or autoimmune disease could be identified, this could allow them to be controlled using vaccines, explains Adrian Hill (Institute of Molecular Medicine, University of Oxford, Oxford, UK). The existing methods for this identification, he says,are mostly indirect. They only work well when a clue to the identity of tbe putative antigen is known. The direct identification methods are very labour intensive and therefore inappropriate for an ‘average’ immunology lab. Hill is interested in this new technology because of its potential to find antigens on infected cells and to use tbis to develop vaccines for diseases as diverse as ADS, TB and malaria. He eagerly awaits the published reports on the system’sperformance. Using less sensitive technology, Hunt and his collaborators have so far identified peptides that stimulate CTLs from melanoma cells, leukaemia cells, several tumours induced by chemical carcinogens in animal models, and bacterial infections in mice. They have also identified HJV epitopes and minor bistocompatibility antigens (these are involved in femals-male tissue rejection). ‘To date,’ enthuses Hunt, ‘more than 25 antigens have been characterized.’ The group is currently trying to identify antigens involved in a number of other cancers and in autoimmune diseases such as diabetes, arthritis and multiple sclerosis. Hunt points out that the new ion-trap mass spectrometer costs approximately $150 000, a third of the price of the conventional triple quadrupole instruments currently in use. Hill is impressed: ‘This is a pretty good price for a mass spectrometer,’ he says.
Elsevier
DavidBradley
Science
Ltd. All rights reserved.
1357 - 4310/97/$17.00
MOLECULAR
MEDICINE
TODAY,
JANUARY
1997
Sensitivepeptide sequenceanalysis: a faster weigh-in for biomolecules A new analytical technique developed by chemists in the USA should speed up the characterization and sequence analysis of minute quantities of peptides, proteins and other biomolecules. This has important implications for several branches of molecular medicine. Donald Hunt (Dept of Chemistry, University of Virginia, Charlottesville, VA, USA), a pioneer in the use of mass spectrometric methods for protein and peptide sequence analysis, has adapted a new liquid chromatography quadrupole (LCQ) ion-trap mass spectrometer from Finnigan MAT Corp. (San Jose, CA, USA) to make it even more sensitive. Hunt’s team was involved in the original conception of the LCQ, although Finmgan holds the patents. He and his team have now replaced tbe LCQ’s standard electrospray ionization source with a novel sheathlessnano-electmspray ionization source to increase its sensitivity. According to Hunt, the modified LCQ coupled with microcapillary high-performance liquid chromatography (HPLC) can be used to sequence as little as lo-‘s mol of a peptide. ‘The technique is 10 000 times more sensitivethan the classical microsequence analysis by Edman degradation,’ he says.This should allow a large number of problems in biochemistry, cell biology and immunology to be solved, because if a peptide sequence is known, the protein can often be identified from its genomic sequence. One such application involves identifying the peptide antigens recognized by cytotoxic T cells (CTLs; see Box 1). ‘This could be the tirst step in the preparation of vaccines that promote immunity against a variety of diseases,’ saysHunt. He and other collaborators (such as the groups of Victor Engelhard and Craig Slingluff, also at the University of Virginia) are using an approach that starts with eluting peptides from diseased cells. The peptides are then separated into fractions using HPLC, and these are assayedfor T-cell-stimulating activity. ‘By combining this T-cell assay with mass spectrometry,’ explains Hunt, ‘it is possible to define the mass of the peptide in the mixture that reconstitutes the epitope (the T-cell stimulatoty portion) recognized by the immune system.’ To tind the sequence of this peptide, the peptides in the biologically active fraction are separated, again using HPLC. The purified peptide is then injected directly into the ion-trap massspectrometer, where individual molecules are ionized and trapped if they fall within a set maximum and minimum mass-to-charge ratio. Trapped peptide ions are then bombarded with fast-moving helium
2
atoms, which randomly break them into smaller charged fragments at their amide bonds. The ion trap stores the fragments so that a much stronger signal than usual is produced in the mass spectrum. ‘This is where the power of this mass spectrometer lies,’ saysteam member Bob Settlage, ‘conventional MS devices look at only one ion at a time, while the ion trap stores all ions before scanning. From the spectrum of massesof these fragments, the researchers can work out the difference between two fragments differing by only a single amino acid. This means that they can defme the mass and thus the identity of the extra residue in the longer of the two fragments. When all the fragments have been analysed, the total sequence of amino acids in the original peptide can be deduced. ‘The ion trap also wins because it can fragment fragments,’ adds Settlage, ‘allowing the deduction of sequences that otherwise would be difficult to determine.’ The most challenging part of this approach is to analyse the minute quantities of immunostimulatory peptides present on the surfaces of diseased cells. These cells might be available in relatively small quantities and are often very expensive to grow up into larger quantities. CTLs are extremely sensitive: they can recognize one copy of a particular peptide per cell. This equates to only 1.5 X IO-t5 mol of peptide on lo9 cells. The microspray-HBLC-ion-trap technology has already been used to sequence lo-t6 mol of a tissue transplantation antigen. The reported results were obtained from 160 000 of a sample of 50 X 10’ cells. Although they haven’t yet repeated this procedure on a smaller number of cells, they expect to get the same results using 10’ cells. The availability of suitable CTLs and the numbers of CTLs required per assaycan also be a limiting factor in this approach, so any
Copyright
01997
economy resulting from increased sensitivity is very useful indeed. If the tiny amounts of peptide antigens produced as a result of viral infection, cancer or autoimmune disease could be identified, this could allow them to be controlled using vaccines, explains Adrian Hill (Institute of Molecular Medicine, University of Oxford, Oxford, UK). The existing methods for this identification, he says,are mostly indirect. They only work well when a clue to the identity of tbe putative antigen is known. The direct identification methods are very labour intensive and therefore inappropriate for an ‘average’ immunology lab. Hill is interested in this new technology because of its potential to find antigens on infected cells and to use tbis to develop vaccines for diseases as diverse as ADS, TB and malaria. He eagerly awaits the published reports on the system’sperformance. Using less sensitive technology, Hunt and his collaborators have so far identified peptides that stimulate CTLs from melanoma cells, leukaemia cells, several tumours induced by chemical carcinogens in animal models, and bacterial infections in mice. They have also identified HJV epitopes and minor bistocompatibility antigens (these are involved in femals-male tissue rejection). ‘To date,’ enthuses Hunt, ‘more than 25 antigens have been characterized.’ The group is currently trying to identify antigens involved in a number of other cancers and in autoimmune diseases such as diabetes, arthritis and multiple sclerosis. Hunt points out that the new ion-trap mass spectrometer costs approximately $150 000, a third of the price of the conventional triple quadrupole instruments currently in use. Hill is impressed: ‘This is a pretty good price for a mass spectrometer,’ he says.
Elsevier
DavidBradley
Science
Ltd. All rights reserved.
1357 - 4310/97/$17.00