Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
  • G01N33/6848—Methods of protein analysis involving mass spectrometry
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2458/00—Labels used in chemical analysis of biological material
  • G01N2458/15—Non-radioactive isotope labels, e.g. for detection by mass spectrometry

Definitions

• the present invention relates to a method for protein protection. More closely, the present invention relates to protection of cysteines in proteins/peptides before they are labelled and analysed, for example by mass spectrometry.
• Cysteines contain thiols, which are among the most reactive groups found in proteins.
• the thiol group can oxidize to form a disulphide (RSSR), a structure that is common in many proteins as internal or external disulphide bridges.
• RSSR disulphide
• Thiols are also avid ligands for heavy metal ions.
• cysteines For protein analysis it is often desired to protect the cysteines, i.e. prevent them from participating in unwanted reactions interfering with the analysis of said proteins. Furthermore, it is usually desired to label the proteins before analysis and in this case it is very important to protect the cysteines to achieve an efficient labelling reaction.
• Oxidation, alkylation, and disulfide exchange are three general approaches used to protect cysteines.
• Alkylation is a common way to block a free cysteine from unwanted side reactions.
• alkylation reagents possible to use for protection of cysteines. Examples are iodoacetamide, acrylamide, maleimide and vinylpyridine.
• iodoacetate or iodoacetamide are used for reaction with thiols to produce stable thioether links.
• Iodoacetamide can sometimes react with methionine residues [1]. They may also react with histidine [2] or tyrosine, but generally only if free thiols are absent.
• Maleimides are excellent reagents for thiol-selective modification. The reaction involves addition of the thiol across the double bond of the maleimide to yield a thioether.
• Maleimides apparently do not react with methionine, histidine or tyrosine. Reaction of maleimides with amines usually requires a higher pH than reaction of maleimides with thiols.
• maleimides hydrolysis of maleimides to a mixture of isomeric nonreactive maleamic acids, which can compete significantly with thiol modification, particularly above pH 8.
• maleimide adducts can hydrolyze, which may cause a significant change in the properties of the conjugate [3], or they can ring-open by nucleophilic reaction with an adjacent amine to yield cross linked products [4].
• Labelling of proteins can be used for a wide range of purposes, e.g. detection, quantification and immobilization.
• One way to introduce labels into proteins is to use some kind of chemical reagent that is reactive towards different functional groups in the proteins.
• N-hydroxy succinimide (NHS) ester modified groups that react readily with lysine residues and the N-terminus of proteins using aqueous physiological conditions.
• N-terminal labelling reagent is the CAFTM labelling reagent for MS.
• Another widely used labelling reagent is ICATTM for cysteine labelling before MS.
• cysteines generally need to be protected before or during labelling and analysis.
• the present invention provides such an improved method for cysteine protection in proteins/peptides to be analysed.
• the present invention relates to a compound giving a highly specific and efficient protection of free thiols in peptides/proteins i.e. it reacts more or less completely with all available cysteines but not with other amino acids present.
• the compound does not interfere with subsequent labelling of the peptide/protein i.e. it is inert towards chemicals other than reducing agents e.g. thiol reagents.
• the compound allows use of different labelling strategies without having to worry about unwanted side-reactions between labelling agents and cysteines. This will permit the subsequent labelling to be performed at rather flexible conditions compared with labelling in the presence of non-modified cysteines.
• the compound of the invention does not interfere with mass spectrometric analysis.
• the cysteine protection is stable and does not add noise into mass spectrometric analyses in any way.
• the invention relates to use of, or method of using, a disulphide- comprising compound to protect cysteines in proteins/peptides from undesired reactions.
• the compound is non-reactive per se.
• the labelling could be any labelling of any amino acid present in said protein/peptide and may be, for example, chemical or enzymatic labelling.
• Labelling before MS is the preferred use but electrophoretic labelling is also envisaged, for example with CyDyeTM DIGE Fluor dyes.
• the use is especially suitable for sensitive MS analysis where it is essential that the Cys-protecting compound is highly selective.
• a further advantage of the protective compound used according to the invention is that it is very stable and resists the harsh labelling conditions.
• the labelling is of other amino acids than Cys.
• Cys can usually not be labelled but with the present invention it is possible to first label one amino acid while Cys is being protected and then deprotect Cys and label Cys with a Cys-labelling agent.
• the aliphatic disulphide is preferably selected from the group that consists of bis-(2- hydroxyethyl) disulphide; bis-(2-hydroxypropyl) disulphide; 3,3- dipropionamidedisulphide and 2,2 A dipyridyl disulphide.
• the compound is polar and not charged.
• a most preferred compound is bis-(2-hydroxyethyl)disulphide, commercially available under the tradename DeStreakTM.
• the labelling may be anywhere in the protein/peptide and can also be at the C- or N- terminal of the peptide or protein.
• the labelling comprises derivatization with any reagent suitable for mass analysis, for example CAFTM.
• a preferred example is labelling of Met with a labelling reagent comprising bromoacetophenone, BAP.
• the use according to the invention may also comprise differential labelling of two samples.
• the samples could be labelled with isotopic variants of the same labelling compound.
• the invention provides a kit for MS or electrophoresis comprising a disulphide-comprising compound as described above and one or more of the following compounds: a labelling agent, control peptides, and buffers.
• the labelling agent may be any labelling agent, such as CAFTM or CyDyeTM.
• the kit comprises DeStreakTM and a BAP-comprising labelling reagent in separate containers.
• the present invention relates to a reagent having a protective action on cysteine residues in peptides or proteins. This protective action is especially useful during chemical labelling of proteins e.g. labelling of amino acids other than cysteine.
• the reagent is non- reactive to other chemicals (except for reducing agents) making it harmless for most protein labelling reagents.
• the reagent is an aliphatic disulphide, preferably bis(2-hydroxyethyl) disulfide (DeStreakTM) and exhibits specific and efficient cysteine protection during labelling of e.g. methionines and the cysteine-adduct does not itself interfere with subsequent labelling reactions.
• DeStreakTM bis(2-hydroxyethyl) disulfide
• the target for the reagent used in the invention is cysteine and the reaction between the reagent and the target is reversible, i.e. it is possible to unblock the cysteines at a later stage if necessary. This means that the reagent does not interfere with, for example, maleimide labelling of cysteines.
• the reversible feature of the reagent can in fact enable specific dual labelling of, for example, lysines and cysteines, where the cross reactivity between lysine and cysteine residues can be minimized.
• thiols in cysteines are protected by use of the thiol- disulphide exchange reaction. This is a reversible reaction which can be described in two steps:
• the rate constants depend on pH and increases with increasing pH.
• the equilibrium constants are usually near unity, therefore, a large excess of thiol reagent must be used to reduce a disulphide or vice versa a large excess of disulphide is required for the oxidation of a thiol .
• the R group can be 2-hydroxyethyl, but a large number of analogous groups can also be used.
• the demand on this group is that it should allow the establishment of the conditions that shift the equilibrium of reaction ⁇ 1 ⁇ to the right side.
• the demand on the co ⁇ -esponding thiol is that it should only contain one -SH group, it should also contain at least one hydrophilic group in order to give the corresponding disulfide a solubility in the reagent solution high enough to allow it to be used in the concentrations required to shift the equilibrium of reaction ⁇ 1 ⁇ to the right side.
• the corresponding disulfide may have a net charge as long as there is no signal suppression during MS analysis, ideally the corresponding disulfide has a zero net charge.
• Example of useful structures are the following: -S-(CH 2 )n-OH with n>2 and -S-(CH 2 )n-CO-NH 2 with n>l, but it is trivial to suggest many other analogous structures, which would meet the requirements on the R group.
• the method for blocking of cysteinyl thiols to prevent unwanted side-reactions during labelling of peptides or proteins was performed as follows: A peptide containing for example cysteine, methionine and lysine was reduced with a non-thiol reducing agent Tris(2-carboxyethyl)phosphine (TCEP), to ensure complete reduction of the cysteines.
• TCEP Tris(2-carboxyethyl)phosphine
• R-SH a large excess of the aliphatic disulphide compound DeStreak
• RlSSRl aliphatic disulphide compound
• the present inventors have found that the use of an aliphatic disulphide dramatically reduced cross reactions from cysteines in a peptide containing methionines, which were to be labelled.
• the experiments showed that it was possible to prevent unwanted side- reactions by including an aliphatic disulphide, such as DeStreakTM, to the samples prior to the labelling reaction.
• cysteine-adduct (Cys-SSR) is stable and non-reactive to the most common protein labelling reagents and the residual unreacted DeStreak does not have to be removed by e.g. desalting.
• Examples of labelling specificity in association with NP (vinyl pyridine) and DeStreak treatment is shown in Tables 3 and 4.
• a peptide containing cysteine, methionine and lysine residues was used. The following protocol was used.
• model peptide was first protected with eitlier 2-Vp or DeStreak and then labelled with BAP.

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• 2004
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• 2005
  • 2005-04-18 WO PCT/SE2005/000553 patent/WO2005106496A1/en active Application Filing

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