WO2005078451A1 - Procede d'analyse de peptides - Google Patents

Procede d'analyse de peptides Download PDF

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Publication number
WO2005078451A1
WO2005078451A1 PCT/SE2005/000187 SE2005000187W WO2005078451A1 WO 2005078451 A1 WO2005078451 A1 WO 2005078451A1 SE 2005000187 W SE2005000187 W SE 2005000187W WO 2005078451 A1 WO2005078451 A1 WO 2005078451A1
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WO
WIPO (PCT)
Prior art keywords
ion exchange
peptides
medium
peptide
exchange medium
Prior art date
Application number
PCT/SE2005/000187
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English (en)
Inventor
Anders Tangen
Henrik Wadensten
Original Assignee
Ge Healthcare Bio-Sciences Ab
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ge Healthcare Bio-Sciences Ab filed Critical Ge Healthcare Bio-Sciences Ab
Publication of WO2005078451A1 publication Critical patent/WO2005078451A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/96—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation using ion-exchange
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials

Definitions

  • the present invention relates to methods for analyzing peptides, such as tryptic peptides, which are modified or derivatized.
  • MALDI mass spectrometry is a method developed for peptide sequencing.
  • MALDI mass spectrometry offers several advantages in the field of mass spectrometry. For example, it provides a higher sensitivity than the conventional electrospray triple quadropole equipment.
  • MALDI mass spectrometry When used in combination with time-of-flight (TOF) mass analyzers, MALDI mass spectrometry is also applicable to higher mass peptides than can be analyzed with triple quadropole equipment.
  • Electrospray ionization is another useful method of introducing samples into a mass spectrometer and is readily interfaced to powerful separation techniques including liquid chromatography (LC) and various forms of capillary electrophoresis (CE). Highly automated analyses are possible when using LC and CE as the sample purification and introduction devices.
  • LC liquid chromatography
  • CE capillary electrophoresis
  • an amino acid sequence can be identified by a derivatization of the N-terminus of a peptide with one or more acidic moieties having pKa values of less than 2 before analysis by mass spectrometry of the analyte, such as with MALDI or electrospray mass spectrometry.
  • the acidic moiety is preferably a sulfonic acid or a disulfonic acid derivative. The derivatives promote a charge-site-initiated cleavage of backbone amide bonds and they enable the selective detection of only a single series of fragment ions comprising the y-ions.
  • An improved method of identifying amino acids as in WO02/095412 comprises the steps of: derivatization in an aqueous solution of the N-terminus of the peptide, or the N-termini of one or more peptides of the peptide, with a least one acidic reagent comprising a sulfonyl moiety coupled to an activated acid moiety to provide one or more peptide derivatives, which reagent exhibits a half-life in aqueous solution of not less than 10 minutes, preferably not less than about 20 minutes and most preferably not less than about 30 minutes at RT; and analyzing at least one such derivative using a mass spectrometric technique to provide a fragmentation pattern; and interpreting the fragmentation pattern obtained.
  • Chemically assisted fragmentation such as the CAF -reagent described in WO 02/095412, is useful for sequencing peptides, such as wild-type, variant and/or synthetic peptides.
  • the method is especially useful for identifying high molecular weight peptides for use e.g. in the biological and pharmaceutical field. More specifically, chemically assisted fragmentation can be used to facilitate biological studies requiring rapid determination of peptide or peptide sequences; to identify post-translational modifications in proteins and to identify amino acid modifications in variant proteins, such as those used in commercial laundry and cleansing products; to aid in the design of oligonucleotide probes for gene cloning; to rapidly characterize products formed in directed evolution studies; in combinatorial and peptide library identification; and in proteomics.
  • the un-modified peptides may also be broken into fragments inside a mass spectrometer which can add significantly to the complexity of the resulting fragmentation spectra, making the spectra difficult to interpret and complicating the analysis and identification of the peptides.
  • the present invention provides methods and systems for analyzing derivatized peptides, such as tryptic peptides, in which unmodified peptides are removed by ion exchange from a solution containing a mixture of modified and un-modified peptides before the solution is analyzed in for example a mass spectrometer.
  • the method of the invention gives better yield than prior art methods by using very low concentrations of organic solvent in the ion exchange eluent, preferably less than 5%, more preferably less than 2.5% and most preferably 0%.
  • Figure 1 is a diagram showing the number of un-modified peptides eluted from an ion exchange column at different salt plug concentrations and at 0% acetonitrile.
  • Figure 2 is a diagram showing tryptic peptides detected vs. % organic phase in mobile phase used during sample injection on the trap column.
  • a method in accordance with the present invention comprises the following steps: one or more sample(s) comprising one or more peptides is derivatised by means of a chemically assisted fragmentation reagent, such as a CAFTM reagent.
  • the derivatization may be a terminal derivatization or a side chain derivatization of the peptide.
  • a negative or positive charge is obtained on the peptide after derivatisation.
  • Any suitable derivatization reagent may be used.
  • the resulting solution contains a mixture of derivatised peptides and unmodified peptides and this is passed through an ion exchange chromatography column.
  • the unmodified peptides are attracted to the ion exchange medium and the derivatised peptides essentially pass straight through the column.
  • the elution from the ion exchange column is performed without organic solvents or with very low amounts of organic solvents, i.e. the eluent comprises less than 5%,. preferably less than 2.5% and most preferably 0% of organic solvents, such as acetonitrile, methanol, ethanol, glycerol, tetrahydrofuran, dichloromethane or combinations thereof.
  • the output of the column containing the derivatised peptides is further separated and analysed, for example inputted into a mass spectrometer and fragmented. The resulting fragmentation spectra are then analyzed.
  • a tryptic digest of Myoglobin was derivatised in an aqueous solution using a CAF MALDI Sequencing kit from Amersham Bioscience AB, Sweden and following the standard procedure in the instruction manual Amersham Biosciences.
  • the resulting solution was then passed through a cation ion exchange column (0.3 x 30 mm BioBasicTM (from Thermo Electron, USA) packed with 5 ⁇ m beads with 300 A pore size) with an eluent devoid of organic solvent.
  • the solution passing straight through the column was fed into a trap column (0.3 x 5 mm, PepMapTM (LC Packings, USA) C18, 5 ⁇ m bead size, 100 A pore size, the trap column was washed with a wash volume of 50 ⁇ l, and then the trapped peptides were eluted to a reversed phase chromatography column (0.075 x 150 mm PepMapTM (LC Packings, USA) , C18 media, 3 ⁇ m bead size, 100 A pore size).
  • a reversed phase chromatography column 0.075 x 150 mm PepMapTM (LC Packings, USA) , C18 media, 3 ⁇ m bead size, 100 A pore size).
  • the flow-through from the ion exchange column was inputted to the mass spectrometer and this resulted in a very distinct mass spectrum.
  • Four CAF-modifed peptides were detected at mass over charge (m/z) positions 871.8, 1449.1, 778.9 and 884.1. As the flow through contains mainly derivatives, the signal to noise ratio of the peptides detected at these positions was very high.
  • the fraction eluted from the same ion exchange column with a 400 mM salt plug was analyzed in the same mass spectrometer.
  • the result from this comparative example is a very complex mass spectrum. Accordingly, when more organic solvent is used in the eluent, as is conventional within prior art to minimize hydrophobic interacation, more peptides will be present in the flow-through, h this spectrum the back ground noise, some of which is assumed to be due to the undesired fragmentation of unmodified peptides, was considerably higher and the signal to noise ratio of the signal at the mass over charge (m/z) positions 871.8, 1449.1, 778.9 and 884.1 was very low.
  • the undesired peptides detected from the 400 mM salt eluate would have been present with the derivatives and would have adversely affected the signal to noise ratio when the sample was analyzed in the mass spectrometer.
  • the purpose of the ion exchange column was to retain un-derivatised or un-modified peptides and the purpose of the reversed phase column was to separate the derivatised peptides eluted from the ion exchange column.
  • FIG. 1 shows a graph in which the number of peptides (N) eluted from the ion exchange column are plotted against the concentration (C) of NH 4 C1 salt plugs (each of 20 ⁇ l) passed through the column after a solution containing 6 unmodified tryptic digested proteins (Cytochrome c, Lysozyme, Alcohol dehydrogenase, Bovine serum albumin, Apo-transferrin, ⁇ -Galactosidase in "Protein Mixture Digest (lyophilized)" P/N 161088 from LC Packings, USA) has been fed through the column.
  • C concentration
  • the elution from the ion exchange column was made with a buffer of 0.4% HAc and 0.005% HFBA in water and, importantly, no organic solvent. Subsequently, the indicated concentrations of NH 4 C1 (0, 40, 100 and 400 nM) and no organic solvent were added.

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  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)

Abstract

L'invention concerne un procédé d'analyse de peptides. Plus précisément, le procédé d'analyse de peptides dans un ou plusieurs échantillons comprend les étapes consistant à dérivatiser les peptides au moyen d'un réactif de fragmentation à assistance chimique, aux fins de production de solutions d'échantillons renfermant un mélange de peptides non dérivatisés et d'un ou de plusieurs dérivés; à faire circuler lesdites solutions dans un milieu échangeur d'ions et à éluer lesdits échantillons sans solvants organiques, des peptides non dérivatisés étant ainsi capturés au moyen dudit milieu et des dérivés passant dans le milieu; et à analyser au moins un dérivé dans la solution d'échantillon prélevé du milieu échangeur d'ions. RPC et MS sont exécutés, de préférence, après l'étape d'échange d'ions.
PCT/SE2005/000187 2004-02-13 2005-02-11 Procede d'analyse de peptides WO2005078451A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE0400358A SE0400358D0 (sv) 2004-02-13 2004-02-13 Peptide identification
SE0400358-8 2004-02-13

Publications (1)

Publication Number Publication Date
WO2005078451A1 true WO2005078451A1 (fr) 2005-08-25

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SE (1) SE0400358D0 (fr)
WO (1) WO2005078451A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8647880B2 (en) 2010-01-25 2014-02-11 Rudjer Bosckovic Institute Mass spectrometry-based protein identification method with selective N-terminus derivatization

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002095412A2 (fr) * 2001-05-23 2002-11-28 Amersham Biosciences Ab Fragmentation peptidique
WO2004015391A2 (fr) * 2002-08-12 2004-02-19 Cornell Research Foundation, Inc. Identification de proteines par spectrometrie de masse
WO2004097427A1 (fr) * 2003-05-02 2004-11-11 Ludwig Institute For Cancer Research Methodes d'analyse de peptide par spectrometrie de masse

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002095412A2 (fr) * 2001-05-23 2002-11-28 Amersham Biosciences Ab Fragmentation peptidique
WO2004015391A2 (fr) * 2002-08-12 2004-02-19 Cornell Research Foundation, Inc. Identification de proteines par spectrometrie de masse
WO2004097427A1 (fr) * 2003-05-02 2004-11-11 Ludwig Institute For Cancer Research Methodes d'analyse de peptide par spectrometrie de masse

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BETANCOURT L. ET AL: "Selective isolation and identification of N-terminal blocked peptides from tryptic protein digests.", J.PEPTIDE RES., vol. 57, 2001, pages 345 - 353, XP002366114, DOI: doi:10.1034/j.1399-3011.2001.00825.x *
WU S.L. ET AL: "Targeted Proteomics of Low-Level Proteins in Human Plasma by LC/MSn: Using Human Growth Hormone as a Model System.", JOURNAL OF PROTEOME RESEARCH., vol. 1, 2002, pages 459 - 465, XP002982668, DOI: doi:10.1021/pr025537l *
YOUNG P. ET AL: "Optimization of high-performance liquid chromatographic peptide separations with alternative mobile and stationary phases.", JOURNAL OF CHROMATOGRAPHY., vol. 512, 1990, pages 273 - 281, XP026475745, DOI: doi:10.1016/S0021-9673(01)89494-2 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8647880B2 (en) 2010-01-25 2014-02-11 Rudjer Bosckovic Institute Mass spectrometry-based protein identification method with selective N-terminus derivatization

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