WO2011071359A2 - Covalent modification of proteins for the instantaneous visualisation thereof and subsequent characterisation through mass spectrometry - Google Patents

Covalent modification of proteins for the instantaneous visualisation thereof and subsequent characterisation through mass spectrometry Download PDF

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WO2011071359A2
WO2011071359A2 PCT/MX2010/000144 MX2010000144W WO2011071359A2 WO 2011071359 A2 WO2011071359 A2 WO 2011071359A2 MX 2010000144 W MX2010000144 W MX 2010000144W WO 2011071359 A2 WO2011071359 A2 WO 2011071359A2
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solution
proteins
protein
sample
covalent modification
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PCT/MX2010/000144
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WO2011071359A3 (en
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Robert Winkler
Marco Arnulfo MATA GÓMEZ
Matthew Thomas Yasui
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Instituto Tecnológico y de Estudios Superiores de Monterrey
Cantu Ortiz, Francisco Javier
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/13—Labelling of peptides

Definitions

  • the present invention broadly relates to the chemical modification of proteins. More specifically, it describes methods for chemically modifying a specific site in the protein peptide chains.
  • the invention is a method for carrying out a covalent modification of proteins for instant visualization and subsequent characterization using mass spectrometry.
  • the first step of a protein analysis is generally the separation of the proteins in polyacrylamide gels with sodium dodecyl sulfate (SDS-PAGE) followed by protein staining.
  • SDS-PAGE sodium dodecyl sulfate
  • the bands corresponding to the protein of interest can be cut directly from the gel and analyzed by mass spectrometry.
  • Coomassie blue staining is one of the most frequently used techniques for protein staining in SDS-PAGE gels, due to its high compatibility with mass spectrometry analysis. However, this technique requires several hours. In addition to that, the dye must be removed before preparing the proteins for mass spectrometry analysis, this procedure takes at least one hour [1].
  • Various stains for covalent protein modification have been described in the literature [2], [3] but the subsequent application of these stains in protein characterization by mass spectrometry is not described.
  • Figure 1 Schematic representation of the reaction of adding Uniblue A to an amino group of the protein.
  • FIG. 1 Visualization of an SDS-PAGE gel stained with Uniblue A. Bovine serum albumin (ASB) sample.
  • ASB Bovine serum albumin
  • Lane 1 Molecular marker. Lane 2: ASB sample stained with Uniblue A
  • Figure 2-b Visualization of an SDS-PAGE gel stained with Coomassie blue. ASB sample.
  • Lane 1 Molecular marker.
  • Lane 2 ASB sample stained with Coomassie blue.
  • Figure 3-a Visualization of an SDS-PAGE gel stained with Uniblue A. Sample of Rituximab, a recombinant antibody.
  • Lane 1 Molecular marker.
  • Figure 3-b Visualization of an SDS-PAGE gel stained with Coomassie blue. It shows Rituximab, a recombinant antibody.
  • Lane 1 Molecular marker.
  • FIG. 4 Analysis of samples of Escherichia coli TOP10 cells with and without expression of a recombinant protein.
  • This protein is a fusion protein, malE-lacZa, from the pMAL-c4x vector (New England Biolabs Inc., NEB).
  • the theoretical molecular weight of this protein is 50871.3 Da, which corresponds to the blue line of the molecular marker.
  • Figure 4-a Visualization of an SDS-PAGE gel stained with Uniblue A for the identification of the expression of a recombinant Escherichia coli protein.
  • Lane 1 Molecular marker.
  • Lane 2 Profile of recombinant Escherichia coli proteins stained with Uniblue A.
  • Lane 3 Un stained recombinant Escherichia coli protein profile.
  • Figure 4-b Visualization of an SDS-PAGE gel stained with Coomassie blue for the identification of the expression of a recombinant Escherichia coli protein.
  • Lane 2 Profile of recombinant Escherichia coli proteins stained with Uniblue A and Coomassie blue.
  • Lane 3 Profile of recombinant Escherichia coli proteins stained with Coomassie blue.
  • Figure 4-c Direct comparison between strains of Escherichia coli without and with expression of a fusion protein of 50871.3 Da. Fast staining of 1 minute only with Uniblue A.
  • Lane 1 Molecular Marker Lane 2: Escherichia coli TOP10 / pMAL-c4x protein profile that produces a MalE-lacza fusion protein, staining with Uniblue A
  • Lane 3 Escherichia coli TOP10 protein profile without expression, staining with Uniblue A
  • Figure 5-a Mass spectrum. ASB peptides analyzed by MS MS without modification.
  • Figure 5-b Mass spectrum. ASB peptides analyzed by MS / MS with modification in Usina K (1).
  • Figure 5-c Mass spectrum. Direct comparison between the peptides with Uniblue A, modification (above) and without modification (below).
  • the ions of the N-terminal group (bions) are easier to detect due to the addition of the mass corresponding to Uniblue A (484.0 Da).
  • FIG. 1 Schematic representation of the reason method of this invention and the preparative stages of samples.
  • Figure 7 Schematic representation of the "a”, “b” and “c” (N-terminal) and "x", “y” and “z” (C-terminal) fragments of a peptide.
  • the present invention relates to a method for chemically modifying a protein, particularly by nucleophilic addition of a dye with at least one functional group, to react in a nucleophilic addition with the amino groups of the protein, preferably at the Usina amino acids of the peptide sequence.
  • a dye for example a vinyl group
  • the dye is Uniblue A (see figure 1).
  • the chemical modification of specific sites in the sequence of a protein with the dye demonstrates certain advantages for its visualization on an SDS-PAGE gel.
  • the advantages are directly related to the visualization of proteins in a short period of time, without noticeable modification of their apparent mass in the SDS-PAGE, to subsequently allow their characterization by mass spectrometry, considerably reducing the analysis time. Additionally, the modification of the peptides can help to obtain more significant results in the analysis of the peptides by changes in the properties of the peptide.
  • SDS sodium dodecyl sulfate
  • SDS is an anionic detergent, which is capable of dissolving hydrophobic molecules.
  • the function of the SDS is to denature the proteins causing a change in the structure (primary, secondary, tertiary or quaternary) giving rise to a linear structure.
  • Another function of SDS is to negatively charge proteins. These two functions are important to carry out an adequate separation of the proteins in an SDS-PAGE gel.
  • Coloring solution refers to a dye in solution, where the dye has a functional group to react in a nucleophilic addition with the amino groups of the protein, preferably in the amino acids lysine of the peptide sequence; and optionally it has at least one other ionizable functional group such as sulfate, to give greater solubility to water staining, subsequently to give greater solubility to the peptides and influences their ionization by mass spectrometry.
  • it is Uniblue A, at a concentration of 60 mM, dissolved in buffer solution A.
  • This solution has a pH of 6.8, and consists of a solution of glycerol (20% v / v), Tris HC1 pH 6.8 (200 mM) and 20 mM dithiothreitol (DTT).
  • DTT dithiothreitol
  • Alkylation solution consists of a solution of iodoacetamide (IAA) at a concentration of 550 mM in buffer solution A. Alkylation occurs by the covalent bond between iodoacetamide and the SH groups present in the cistern residues.
  • IAA iodoacetamide
  • ASB lyophilized bovine serum albumin
  • Protein samples to be analyzed must meet the following characteristics.
  • Protein samples to be characterized by the method proposed in this application have been classified as described below.
  • sample of protein in solution in a low concentration and non-compatible buffer solution (say non-compatible buffer solution such as that containing amino groups, eg: Tris).
  • -In samples of proteins in solution in a sufficient concentration and a compatible buffer solution (3) enter stage a) of the proposed method.
  • -In samples in solution in which there is a low concentration of proteins and others have a non-compatible buffer solution (5) optionally:
  • -The proteins can be precipitated using TCA / acetone and subsequently resuspended in buffer solution A (6); This is done by mixing the TCA / acetone (lg TCA / mL acetone, previously cooled to 4 ° C) and the liquid protein sample in a 1: 9 ratio. The mixture is placed in an ice bath at 4 ° C for 1-2 hours. Then, the samples are centrifuged at 14,000 rpm at 4 ° C for 15 minutes.
  • the supernatant is removed and the precipitate is washed with acetone at least three times, then centrifuged at 14,000 rpm for 5 min and the supernatant is removed. Once washed, the precipitate is resuspended in buffer solution A.
  • the non-compatible solution can be eliminated by ultrafiltration and at the same time the proteins are concentrated and subsequently the proteins are recovered with the addition of buffer solution A.
  • the method for covalent protein modification for instant visualization and subsequent characterization using mass spectrometry comprises the following steps: a) Staining a sample containing the protein of interest in solution, with a dye solution with 1 functional group, to react in a nucleophilic addition with the amino groups of the protein, preferably in the Usina amino acids of the peptide sequence; and optionally has at least one other ionizable functional group to give greater solubility to water staining, subsequently give greater solubility to the peptides and influence their ionization by mass spectrometry, the dye solution is Uniblue A dissolved in the buffer solution A in a concentration of 60 mM.
  • the resulting sample solution is heated at 100 ° C for 1 minute, avoiding prolonged exposure of the sample at this temperature because there is a risk of degrading the protein.
  • the chemical reaction also works at lower temperatures, but with a longer incubation time.
  • This step is carried out by adding the alkylation solution in a 10: 1 volume ratio (reduced sample solution: alkylation solution).
  • the time for alkylation is preferably 5 minutes at room temperature.
  • the gel fragments are incubated at a temperature of 60 ° C for 30 minutes, at this stage the trypsin hydrolysis reaction near residues modified with Uniblue A is inhibited, therefore the hydrolysis reaction is carried out mostly in arginine residues obtaining larger proteolytic fragments, achieving an improvement in the coverage of peptide sequencing, it should be noted that at this stage the ions of the N-terminal group preceded by a plant have an additional mass corresponding to Uniblue A (484.0 Da), which facilitates its detection by mass spectrometry, and each lysine modification affects the mass of amino acid fragments in the N or C terminal direction. h) Extraction of the peptides from the gel fragments (14).
  • Another object of the present invention relates to a Kit for instantly visualizing proteins for subsequent characterization using mass spectrometry.
  • the proclaimed Kit comprises the following reagents, stored independently:
  • a buffer solution A has a pH in the range of 8 to 9, preferably it consists of a solution of sodium carbonate at a concentration of 100 mM and a solution of sodium dodecyl sulfate (SDS) at a concentration of 10% ( p / v).
  • SDS sodium dodecyl sulfate
  • the Umblue A dye is dissolved in buffer solution A, at a concentration of 60 mM.
  • This solution has a pH 6.8, and consists of a 20% (v / v) solution of glycerol, 200 mM Tris HC1 and 20 mM dithiothreitol (DTT).
  • Alkylation solution It consists of a solution of iodoacetamide (IAA) at a concentration of 550 mM in buffer solution A.
  • the extracted peptides were recovered by the addition of approximately 20 ⁇ , of a 0.1% (v / v) formic acid solution, the solution with the peptides was transferred in polypropylene vials (FAMOS) to nano LC-MS / MS. Peptide analysis was carried out using a nano LC-ESI-ion trap. The resulting spectrum was evaluated by the use of X! Tandem and OMSSA automatic research algorithms. To quantify the number of covalent modifications, the mass change of + 484.03989 Da (monoisotopic) in the amino acids modified by Uniblue A, was taken into account (see Figure 5-a, 5-b and 5-c).
  • Example 2 Covalent modification of proteins for instant visualization using the Motif Kit of this invention in a sample of a recombinant antibody (Rituximab).
  • buffer solution A For this procedure, an exchange of a non-compatible buffer solution was carried out with buffer solution A.
  • 250 iL of a recombinant Rituximab solution 500mg 50mL were placed in a Amicon® Ultra ultrafiltration tube (Millipore TM) with a capacity of 0.5 mL and a 3000 MWCO membrane (Molecular Weight Cut Off, [Da]).
  • 250 ⁇ - of buffer solution A 250 ⁇ - of buffer solution A.
  • the sample was then centrifuged at 14,000 x g for 30 minutes in a centrifuge for Eppendorf tubes. A sufficient volume of buffer solution A was then added to have a volume of 0.5 mL (maximum capacity of the Eppendorf tube). Repeat this procedure at least five times. After repeating the previous procedure for the last time, Rituximab was resuspended in 125) I of buffer solution A, the final concentration of this solution was 20 mg / mL.
  • Example 3 Covalent modification of proteins for instant visualization using the Kit, this case, in a sample of Escherichia coli, strain TOP10, with and without expression of a recombinant protein with plasmid pMAL-c4x, which codes for a fusion protein .
  • Fusion is given by a maltose binding protein and the lacZa gene.
  • a protein expression control was performed.
  • E. coli strains The propagation of E. coli strains was carried out in 50 mL of TB Overnight Express medium (Novagen Inc.). Additionally, 150 uL of the antibiotic carbencillin was added to the TB medium for the recombinant strain to prevent the growth of other microorganisms. The culture medium was then inoculated with 100 uL of a solution of E. coli. The culture of E. coli was carried out for 16 hours at 37 ° C and stirring of 250 rpm. After incubation, the cell culture medium was separated into 7mL volumes in 15 mL conical tubes and centrifuged at 5,000 xg at a temperature of 4 ° C for 15 minutes.
  • the supernatant was removed and the precipitated E. coli cells, at the bottom of the tube, were resuspended with the addition of 4.5 mL of buffer solution A. Subsequently, the E. coli cell wall rupture was performed to To obtain the proteins present inside the cells, this was done by ultrasonication for 15 minutes. Then, the sample was centrifuged at 10,000 xg for 15 minutes at 4 ° C, and the supernatant was recovered.
  • the proteins present in the supernatant were precipitated by the addition of 0.5 mL of a TCA / acetone solution (lg TCA / mL of acetone) in a ratio of 9: 1 volumes (Supernatant: TCA / acetone solution). This procedure was performed at a temperature of 4 ° C for 1-2 hours. After precipitation, the samples were centrifuged at 10,000 x g for 15 minutes at 4 ° C. The supernatant was discarded and the precipitated proteins were washed with acetone (90%) at least three times (centrifuge at 10,000 x g for 15 minutes between each wash). Finally, the excess acetone was removed and the proteins were reconstituted in 100 uL of buffer solution A.

Abstract

The invention described discloses the covalent modification of proteins for the instantaneous visualisation and subsequent characterisation thereof. The chemical mechanism employed is based on nucleophilic addition. The mechanism of the chemical reaction is explained, together with the bonding of the latter to specific aminoacid residues. Identification and characterisation of stained proteins employing mass spectrometry may be carried out by virtue of changes in mass of the residues modified by Uniblue A. A significant advantage of the staining employed in this invention lies in it having shown high compatibility with mass spectrometry analysis, it being unnecessary in addition to remove the colourant from proteins following staining as in the case of Coomassie or silver staining. Moreover, the derivatisation of amino acids gives rise to a change in the proteolytic hydrolysis patterns which may increase peptide sequencing coverage. Furthermore, improvement in the sequencing coverage of MS/MS analysis may be achieved by taking into account the changes in mass of the terminal amino groups of the derivatised amino acids. Additionally, a shorter procedure for protein identification is presented. Using the methods proposed for staining and analysis of proteins, the time required for protein analysis may be substantially reduced whilst the quality of the analysis may be improved.

Description

MODIFICACIÓN COV ALENTE DE PROTEÍNAS PARA SU VISUALIZACIÓN INSTANTANEA Y POSTERIOR CARACTERIZACIÓN MEDIANTE ESPECTROMETRÍA DE MASAS. DESCRIPCIÓN CAMPO DE LA INVENCIÓN  MODIFICATION COV ALENTE PROTEINS FOR INSTANT AND REAR VIEW CHARACTERIZATION THROUGH MASS SPECTROMETRY. DESCRIPTION FIELD OF THE INVENTION
La presente invención se refiere ampliamente a la modificación química de proteínas. De manera más específica, describe métodos para modificar químicamente un sitio específico en las cadenas peptídicas de las proteínas.  The present invention broadly relates to the chemical modification of proteins. More specifically, it describes methods for chemically modifying a specific site in the protein peptide chains.
OBJETO DE LA INVENCIÓN OBJECT OF THE INVENTION
La invención es un método para llevar a cabo una modificación covalente de proteínas para una visualización instantánea y su posterior caracterización usando espectrometría de masas.  The invention is a method for carrying out a covalent modification of proteins for instant visualization and subsequent characterization using mass spectrometry.
ANTECEDENTES BACKGROUND
El primer paso de un análisis de proteínas, es generalmente, la separación de las proteínas en geles de poliacrilamida con dodecil sulfato de sodio (SDS-PAGE) seguido de la tinción de las proteínas. Las bandas correspondientes a la proteína de interés pueden ser cortadas directamente del gel y analizadas por espectrometría de masas. La tinción con azul de Coomassie, es una de las técnicas más frecuentemente utilizadas para la tinción de proteínas en geles de SDS-PAGE, debido a su alta compatibilidad con el análisis de espectrometría de masas. Sin embargo, esta técnica requiere de varias horas. Además de que, el colorante debe ser removido antes de preparar las proteínas para el análisis de espectrometría de masas, este procedimiento necesita al menos una hora [1]. En la literatura se han descrito diversas tinciones para la modificación covalente de proteínas [2],[3] pero la subsecuente aplicación de estas tinciones en la caracterización de proteínas por espectrometría de masas no es descrita. BREVE DESCRD7CIÓN DE LAS FIGURAS  The first step of a protein analysis is generally the separation of the proteins in polyacrylamide gels with sodium dodecyl sulfate (SDS-PAGE) followed by protein staining. The bands corresponding to the protein of interest can be cut directly from the gel and analyzed by mass spectrometry. Coomassie blue staining is one of the most frequently used techniques for protein staining in SDS-PAGE gels, due to its high compatibility with mass spectrometry analysis. However, this technique requires several hours. In addition to that, the dye must be removed before preparing the proteins for mass spectrometry analysis, this procedure takes at least one hour [1]. Various stains for covalent protein modification have been described in the literature [2], [3] but the subsequent application of these stains in protein characterization by mass spectrometry is not described. BRIEF DESCRD7TION OF THE FIGURES
Figura 1. Representación esquemática de la reacción de adición de Uniblue A a un grupo amino de la proteína.  Figure 1. Schematic representation of the reaction of adding Uniblue A to an amino group of the protein.
Figura 2-a. Visualización de un gel de SDS-PAGE teñido con Uniblue A. Muestra de Albúmina de suero bovino (ASB).  Figure 2-a. Visualization of an SDS-PAGE gel stained with Uniblue A. Bovine serum albumin (ASB) sample.
Carril 1: Marcador molecular. Carril 2: Muestra de ASB teñido con Uniblue A Lane 1: Molecular marker. Lane 2: ASB sample stained with Uniblue A
Figura 2-b. Visualización de un gel de SDS-PAGE teñido con azul de Coomassie. Muestra de ASB.  Figure 2-b. Visualization of an SDS-PAGE gel stained with Coomassie blue. ASB sample.
Carril 1 : Marcador molecular. Lane 1: Molecular marker.
Carril 2: Muestra de ASB teñida con azul de Coomassie. Lane 2: ASB sample stained with Coomassie blue.
Figura 3-a. Visualización de un gel de SDS-PAGE teñido con Uniblue A. Muestra de Rituximab, un anticuerpo recombinante.  Figure 3-a. Visualization of an SDS-PAGE gel stained with Uniblue A. Sample of Rituximab, a recombinant antibody.
Carril 1: Marcador molecular. Lane 1: Molecular marker.
Carril 2: Rituximab teñido con Uniblue A. Lane 2: Rituximab stained with Uniblue A.
Figura 3-b. Visualización de un gel de SDS-PAGE teñido con azul de Coomassie. Muestra Rituximab, un anticuerpo recombinante. Figure 3-b. Visualization of an SDS-PAGE gel stained with Coomassie blue. It shows Rituximab, a recombinant antibody.
Carril 1: Marcador molecular. Lane 1: Molecular marker.
Carril 2: Rituximab teñido con azul de Coomassie. Lane 2: Rituximab stained with Coomassie blue.
Figura 4. Análisis de muestras de células de Escherichia coli TOP10 con y sin expresión de una proteína recombinante. Esta proteína, es una proteína de fusión, malE-lacZa, proveniente del vector pMAL-c4x (New England Biolabs Inc., NEB). El peso molecular teórico de esta proteína es de 50871.3 Da, que corresponde a la línea azul del marcador molecular.  Figure 4. Analysis of samples of Escherichia coli TOP10 cells with and without expression of a recombinant protein. This protein is a fusion protein, malE-lacZa, from the pMAL-c4x vector (New England Biolabs Inc., NEB). The theoretical molecular weight of this protein is 50871.3 Da, which corresponds to the blue line of the molecular marker.
Figura 4-a. Visualización de un gel de SDS-PAGE teñido con Uniblue A para la identificación de la expresión de una proteína recombinante de Escherichia coli.  Figure 4-a. Visualization of an SDS-PAGE gel stained with Uniblue A for the identification of the expression of a recombinant Escherichia coli protein.
Carril 1 : Marcador molecular. Lane 1: Molecular marker.
Carril 2: Perfil de proteínas de Escherichia coli recombinante teñidas con Uniblue A.  Lane 2: Profile of recombinant Escherichia coli proteins stained with Uniblue A.
Carril 3: Perfil de proteína de Escherichia coli recombinante sin teñir. Lane 3: Un stained recombinant Escherichia coli protein profile.
Figura 4-b. Visualización de un gel de SDS-PAGE teñido con azul de Coomassie para la identificación de la expresión de una proteína recombinante de Escherichia coli. Figure 4-b. Visualization of an SDS-PAGE gel stained with Coomassie blue for the identification of the expression of a recombinant Escherichia coli protein.
Carril 1 : Marcador molecular Lane 1: Molecular Marker
Carril 2: Perfil de proteínas de Escherichia coli recombinante teñidas con Uniblue A y azul de Coomassie.  Lane 2: Profile of recombinant Escherichia coli proteins stained with Uniblue A and Coomassie blue.
Carril 3: Perfil de proteínas de Escherichia coli recombinante teñidas con azul de Coomassie.  Lane 3: Profile of recombinant Escherichia coli proteins stained with Coomassie blue.
Figura 4-c. Comparación directa entre las cepas de Escherichia coli sin y con expresión de una proteína de fusión de 50871.3 Da. Tinción rápida de 1 minuto solamente con Uniblue A.  Figure 4-c. Direct comparison between strains of Escherichia coli without and with expression of a fusion protein of 50871.3 Da. Fast staining of 1 minute only with Uniblue A.
Carril 1: Marcador molecular Carril 2: Perfil de proteínas de Escherichia coli TOP10/pMAL-c4x que produce una proteína fusión MalE-lacza, tinción con Uniblue A Lane 1: Molecular Marker Lane 2: Escherichia coli TOP10 / pMAL-c4x protein profile that produces a MalE-lacza fusion protein, staining with Uniblue A
Carril 3: Perfil de proteínas de Escherichia coli TOP10 sin expresión, tinción con Uniblue A  Lane 3: Escherichia coli TOP10 protein profile without expression, staining with Uniblue A
Figura 5-a. Espectro de masas. Péptidos de ASB analizados por MS MS sin modificación. Figura 5-b. Espectro de masas. Péptidos de ASB analizados por MS/MS con modificación en Usina K (1). Figure 5-a. Mass spectrum. ASB peptides analyzed by MS MS without modification. Figure 5-b. Mass spectrum. ASB peptides analyzed by MS / MS with modification in Usina K (1).
Figura 5-c. Espectro de masas. Comparación directa entre los péptidos con Uniblue A, modificación (arriba) y sin modificación (abajo). Los iones del grupo N-terminal (b iones) son más fáciles de detectar debido a la adición de la masa correspondiente al Uniblue A (484.0 Da).  Figure 5-c. Mass spectrum. Direct comparison between the peptides with Uniblue A, modification (above) and without modification (below). The ions of the N-terminal group (bions) are easier to detect due to the addition of the mass corresponding to Uniblue A (484.0 Da).
Figura 6. Representación esquemática del método motivo de esta invención y las etapas preparativas de muestras.  Figure 6. Schematic representation of the reason method of this invention and the preparative stages of samples.
Figura 7. Representación esquemática de los fragmentos "a", "b" y "c" (N-terminal) y "x", "y" y "z" (C-terminal) de un péptido.  Figure 7. Schematic representation of the "a", "b" and "c" (N-terminal) and "x", "y" and "z" (C-terminal) fragments of a peptide.
DESCRIPCIÓN DETALLADA DE LA INVENCIÓN DETAILED DESCRIPTION OF THE INVENTION
La presente invención refiere un método para modificar químicamente una proteína, particularmente mediante la adición nucleofílica de un colorante con al menos un grupo funcional, para reaccionar en una adición nucleofílica con los grupos amino de la proteína, preferentemente en los aminoácidos Usina de la secuencia peptídica; (por ejemplo un grupo vinil), y opcionalmente tiene al menos otro grupo funcional ionizable para dar mayor solubilidad a la tinción en agua, posteriormente dar mayor solubilidad a los péptidos e influye en su ionización por espectrometría de masas (por ejemplo un grupo sulfato); preferentemente el colorante es Uniblue A (ver figura 1). La modificación química de sitios específicos en la secuencia de una proteína con el colorante, demuestra ciertas ventajas para su visualización en un gel de SDS-PAGE. Las ventajas están directamente relacionadas con la visualización de las proteínas en un corto período de tiempo, sin modificación notable de su masa aparente en el SDS-PAGE, para posteriormente permitir su caracterización mediante espectrometría de masas, disminuyendo considerablemente el tiempo de análisis. Adicionalmente, la modificación de los péptidos puede ayudar para obtener resultados más significantes en el análisis de los péptidos por cambios de las propiedades del péptido.  The present invention relates to a method for chemically modifying a protein, particularly by nucleophilic addition of a dye with at least one functional group, to react in a nucleophilic addition with the amino groups of the protein, preferably at the Usina amino acids of the peptide sequence. ; (for example a vinyl group), and optionally has at least one other ionizable functional group to give greater solubility to water staining, subsequently give greater solubility to the peptides and influences their ionization by mass spectrometry (for example a sulfate group) ; preferably the dye is Uniblue A (see figure 1). The chemical modification of specific sites in the sequence of a protein with the dye demonstrates certain advantages for its visualization on an SDS-PAGE gel. The advantages are directly related to the visualization of proteins in a short period of time, without noticeable modification of their apparent mass in the SDS-PAGE, to subsequently allow their characterization by mass spectrometry, considerably reducing the analysis time. Additionally, the modification of the peptides can help to obtain more significant results in the analysis of the peptides by changes in the properties of the peptide.
Para llevar a cabo el método propuesto se requieren las siguientes soluciones:  The following solutions are required to carry out the proposed method:
a) Solución amortiguadora A. El rango de pH de esta solución es de 8-9.  a) Buffer solution A. The pH range of this solution is 8-9.
Preferentemente consiste de una solución de carbonato de sodio (100 mM) y dodecilsulfato sódico (SDS) al 10% (p/v) en solución con agua bidestilada. El SDS es un detergente aniónico, el cual es capaz de disolver moléculas hidrófobas. La función del SDS es desnaturalizar las proteínas originando un cambio en la estructura (primaria, secundaria, terciaria o cuaternaria) dando lugar a una estructura lineal. Otra de las funciones del SDS es cargar negativamente las proteínas. Estas dos funciones son importantes para llevar a cabo una adecuada separación de las proteínas en un gel de SDS-PAGE. It preferably consists of a solution of sodium carbonate (100 mM) and 10% (w / v) sodium dodecyl sulfate (SDS) in solution with double distilled water. SDS is an anionic detergent, which is capable of dissolving hydrophobic molecules. The function of the SDS is to denature the proteins causing a change in the structure (primary, secondary, tertiary or quaternary) giving rise to a linear structure. Another function of SDS is to negatively charge proteins. These two functions are important to carry out an adequate separation of the proteins in an SDS-PAGE gel.
b) Solución colorante. Se refiere a un colorante en solución, donde el colorante tiene un grupo funcional para reaccionar en una adición nucleofilica con los grupos amino de la proteína, preferentemente en los aminoácidos lisina de la secuencia peptídica; y opcionalmente tiene al menos otro grupo funcional ionizable como el sulfato, para dar mayor solubilidad a la tinción en agua, posteriormente dar mayor solubilidad a los péptidos e influye en su ionización por espectrometría de masas. Preferentemente, es Uniblue A, a una concentración de 60 mM, disuelto en la solución amortiguadora A.  b) Coloring solution. It refers to a dye in solution, where the dye has a functional group to react in a nucleophilic addition with the amino groups of the protein, preferably in the amino acids lysine of the peptide sequence; and optionally it has at least one other ionizable functional group such as sulfate, to give greater solubility to water staining, subsequently to give greater solubility to the peptides and influences their ionization by mass spectrometry. Preferably, it is Uniblue A, at a concentration of 60 mM, dissolved in buffer solution A.
c) Solución reductora. Esta solución tiene un pH de 6.8, y consiste de una solución de glicerol (20% v/v), Tris HC1 pH 6.8 (200 mM) y ditiotreitol (DTT) 20 mM. La reducción de un enlace disulfuro por DTT, se lleva a cabo por dos reacciones secuenciales, en las cuales se intercambia un grupo sulfidrilo (— SH) presente en el enlace disulfüro (unión covalente entre dos grupos— SH presentes en residuos de cisterna).  c) Reducing solution. This solution has a pH of 6.8, and consists of a solution of glycerol (20% v / v), Tris HC1 pH 6.8 (200 mM) and 20 mM dithiothreitol (DTT). The reduction of a disulfide bond by DTT is carried out by two sequential reactions, in which a sulphydryl group (-SH) present in the disulphide bond is exchanged (covalent bonding between two groups-SH present in cistern residues).
d) Solución de alquilacíón. Consiste en una solución de iodoacetamida (IAA) a una concentración de 550 mM en solución amortiguadora A. La alquiliación se da por la unión covalente entre la iodoacetamida y los grupos— SH presentes en los residuos de cisterna.  d) Alkylation solution. It consists of a solution of iodoacetamide (IAA) at a concentration of 550 mM in buffer solution A. Alkylation occurs by the covalent bond between iodoacetamide and the SH groups present in the cistern residues.
e) Control positivo. 3 mg de albúmina de suero bovino (ASB) liofilizada son reconstituidos en 1 mL de solución amortiguadora A. Una de las razones por la cual se eligió la ASB, es porque es una proteína muy bien caracterizada y estudiada, lo que permite utilizarla como modelo para llevar a cabo diversos experimentos, además de su disponibilidad en el mercado a un bajo costo.  e) Positive control. 3 mg of lyophilized bovine serum albumin (ASB) are reconstituted in 1 mL of buffer solution A. One of the reasons why ASB was chosen is because it is a very well characterized and studied protein, which allows it to be used as a model to carry out various experiments, in addition to their availability in the market at a low cost.
Las muestras proteicas a analizar deben cumplir con las siguientes características. Protein samples to be analyzed must meet the following characteristics.
a) Estar en solución con al menos 50% de solución amortiguadora A.  a) Be in solution with at least 50% buffer solution A.
b) Tener una concentración de proteína en al menos 100 mg mL de solución. c) Carecer de compuestos aminos que puedan obstruir o reaccionar con el colorante Uniblue A. b) Have a protein concentration in at least 100 mg mL of solution. c) Lack amino compounds that can obstruct or react with Uniblue A dye.
Las muestras proteicas a caracterizar por el método propuesto en esta solicitud, se han clasificado como a continuación se describe. Protein samples to be characterized by the method proposed in this application have been classified as described below.
a) Muestra de proteína seca (liofilizada).  a) Dry protein sample (lyophilized).
b) Muestra de proteínas en solución en una concentración suficiente y una solución amortiguadora compatible.  b) Sample of proteins in solution in a sufficient concentration and a compatible buffer solution.
c) Muestra de proteína en solución en una baja concentración y solución amortiguadora no compatible, (dígase de solución amortiguadora no compatible como aquella que contiene grupos amino, p.ej.: Tris).  c) Sample of protein in solution in a low concentration and non-compatible buffer solution, (say non-compatible buffer solution such as that containing amino groups, eg: Tris).
Dependiendo del origen de la muestra que contiene la proteína, se presenta un protocolo (ver figura 6) para la preparación de la muestra, de tal modo que: Depending on the origin of the sample containing the protein, a protocol is presented (see figure 6) for the preparation of the sample, so that:
- En muestras de proteína seca (1), se disuelve (2) la muestra en una solución amortiguadora A.  - In samples of dry protein (1), the sample is dissolved (2) in a buffer solution A.
-En muestras de proteínas en solución en una concentración suficiente y una solución amortiguadora compatible (3), ingresan a la etapa a) del método propuesto. -En muestras en solución en las cuales existe una baja concentración de proteínas y a demás presentan una solución amortiguadora no compatible (5), opcionalmente: -Las proteínas pueden ser precipitadas usando TCA/acetona y posteriormente resuspendidas en solución amortiguadora A (6); esto se lleva a cabo mezclando el TCA/acetona (lg TCA/mL acetona, previamente enfriada a 4 °C) y la muestra líquida de proteína en relación 1:9. La mezcla se coloca en un baño con hielo a 4 °C por 1-2 horas. Después, las muestras son centrifugadas a 14,000 rpm a 4 °C por 15 minutos. El sobrenadante es removido y el precipitado es lavado con acetona al menos tres veces, después es centrifugado a 14,000 rpm por 5 min y el sobrenadante es eliminado. Una vez lavado, el precipitado es resuspendido en la solución amortiguadora A.  -In samples of proteins in solution in a sufficient concentration and a compatible buffer solution (3), enter stage a) of the proposed method. -In samples in solution in which there is a low concentration of proteins and others have a non-compatible buffer solution (5), optionally: -The proteins can be precipitated using TCA / acetone and subsequently resuspended in buffer solution A (6); This is done by mixing the TCA / acetone (lg TCA / mL acetone, previously cooled to 4 ° C) and the liquid protein sample in a 1: 9 ratio. The mixture is placed in an ice bath at 4 ° C for 1-2 hours. Then, the samples are centrifuged at 14,000 rpm at 4 ° C for 15 minutes. The supernatant is removed and the precipitate is washed with acetone at least three times, then centrifuged at 14,000 rpm for 5 min and the supernatant is removed. Once washed, the precipitate is resuspended in buffer solution A.
-La solución no compatible puede ser eliminada mediante ultrafiltración y al mismo tiempo las proteínas son concentradas y posteriormente las proteínas son recuperadas con la adición de solución amortiguadora A.  -The non-compatible solution can be eliminated by ultrafiltration and at the same time the proteins are concentrated and subsequently the proteins are recovered with the addition of buffer solution A.
El método para la modificación covalente de proteínas para su visualización instantánea y posterior caracterización usando espectrometría de masas comprende las siguientes etapas: a) Teñir una muestra que contiene la proteína de interés en solución, con una solución colorante con 1 grupo funcional, para reaccionar en una adición nucleofilica con los grupos amino de la proteína, preferentemente en los aminoácidos Usina de la secuencia peptídica; y opcionalmente tiene al menos otro grupo funcional ionizable para dar mayor solubilidad a la tinción en agua, posteriormente dar mayor solubilidad a los péptidos e influye su ionización por espectrometría de masas, la solución colorante es Uniblue A disuelto en la solución amortiguadora A en una concentración de 60 mM. La solución muestra resultante es calentada a 100°C durante 1 minuto, evitando prolongar la exposición de la muestra a esta temperatura debido a que se corre el riesgo de degradar la proteína. La reacción química también funciona con temperaturas más bajas, pero con un tiempo de incubación más prolongado. b) Reducción de la proteína (8). Esto se realiza mediante la adición de la solución reductora a la solución muestra obtenida en la Etapa a, en una relación de volúmenes 1: 1 (solución muestra : solución reductora) preferentemente y la posterior incubación en un rango comprendido entre 90°C a 100°C durante un tiempo de al menos 1 minuto; la solución obtenida se le denomina solución muestra reducida. c) Alquilación de los grupos sulfhidrilo (— SH) reducidos (9), presentes en los residuos de cisterna. Esta etapa se lleva a cabo agregando la solución de alquilación en una relación de volúmenes 10:1 (solución muestra reducida: solución de alquilación). El tiempo para la alquilación es preferentemente de 5 minutos a una temperatura ambiente. d) Separación de las proteínas en un gel de poliacrilamida-dodecil sulfato de sodio (SDS-PAGE) (10). La separación se llevó a cabo de acuerdo al protocolo estándar descrito por Sambrook 2001, aplicando una corriente eléctrica de 150 V por 45 minutos. Para este caso la separación de las proteínas se llevó a cabo en geles de poliacrilamida al 10 y 12.5% (aunque la concentración de poliacrilamida usada en los geles depende del peso molecular de las proteínas de interés a separar). e) Cortar las bandas de proteína del gel de SDS-PAGE (11). Esto se realiza cortando la región donde se localiza la banda que corresponde a la proteína de interés en el gel. Posteriormente las bandas con la proteína de interés se cortan en fragmentos más pequeños (lmm x lmm). f) Eliminar el exceso de agua presente en los fragmentos de gel además de fijar las proteínas (12), esto se lleva a cabo Fijando y deshidrando los fragmentos de gel. Esto se realiza por la adición directa de acetonitrilo a los fragmentos de gel hasta cubrirlos e incubando por 5 minutos a temperatura ambiente, posteriormente el exceso de acetonitrilo es removido de las muestras, y estas son secadas mediante centrifugación al vacio por 10 minutos. Este etapa es recomendable para disminuir los efectos del acetonitrilo sobre la tripsina cuando se realiza la digestión. g) Digestión tríptica de las proteínas presentes en los fragmentos de gel fijados y deshidratados (13). Esto se realiza mediante la adición de una solución de tripsina (V511A Promega, 10
Figure imgf000009_0001
en NH4HCO3, 25 mM) suficiente para cubrir los fragmentos de gel previamente fijados y deshidratados. Posteriormente, los fragmentos de gel son incubadas a una temperatura de 60°C durante 30 minutos, en esta etapa la reacción de hidrólisis por tripsina cerca de residuos modificados con Uniblue A es inhibida, por lo tanto la reacción de hidrólisis se realiza en mayor parte en los residuos de arginina obteniendo fragmentos proteolíticos más grandes, logrando un mejoramiento en la cobertura de la secuenciación de péptidos, cabe señalar que en esta etapa los iones del grupo N-terminal precedidos por una Usina tienen una masa adicional correspondiente al Uniblue A (484.0 Da), lo que facilita su detección mediante espectrometría de masas, y cada modificación de lisina afecta la masa de los fragmentos de aminoácidos en dirección N o C terminal. h) Extracción de los péptidos de los fragmentos de gel (14). Esto se lleva a cabo por la adición de una solución de acetonitrilo/ácido trifluoroacético 0.1% (v/v) en relación 1:1 a los fragmentos de gel seguido de la incubación a 60 °C por 15 minutos. Para la obtención de una mayor cobertura de la secuencia de péptidos se recomienda usar tiempos de extracción prolongados. Después de la extracción es importante transferir la solución de extracción a un tubo nuevo. i) Secar los péptidos (15), esto se lleva a cabo eliminando el exceso de solución de acetonitrilo/ácido trifluoroacético por centrifugación a vacío hasta obtener los péptidos secos. Preparación y análisis de los péptidos extraídos mediante espectrometría de masas (16). Los péptidos extraídos son recuperados por la adición de aproximadamente 20 μΐ,. de una solución de ácido fórmico 0.1% (v/v), la solución con los péptidos es transferida en viales de polipropileno (FAMOS) para nano LC-MS MS. El análisis de los péptidos puede ser realizado, p. ej. en un nano LC-ESI- trampa de iones. El espectro resultante puede ser evaluado usando algoritmos de investigación automática como el X!Tandem o OMSSA. Para cuantificar el número de modificaciones covalentes, se toman en cuenta los cambios de masa de + 484.03989 Da (monoisotópico) para Uniblue A. The method for covalent protein modification for instant visualization and subsequent characterization using mass spectrometry comprises the following steps: a) Staining a sample containing the protein of interest in solution, with a dye solution with 1 functional group, to react in a nucleophilic addition with the amino groups of the protein, preferably in the Usina amino acids of the peptide sequence; and optionally has at least one other ionizable functional group to give greater solubility to water staining, subsequently give greater solubility to the peptides and influence their ionization by mass spectrometry, the dye solution is Uniblue A dissolved in the buffer solution A in a concentration of 60 mM. The resulting sample solution is heated at 100 ° C for 1 minute, avoiding prolonged exposure of the sample at this temperature because there is a risk of degrading the protein. The chemical reaction also works at lower temperatures, but with a longer incubation time. b) Protein reduction (8). This is done by adding the reducing solution to the sample solution obtained in Step a, in a ratio of 1: 1 volumes (sample solution: reducing solution) preferably and subsequent incubation in a range between 90 ° C to 100 ° C for a time of at least 1 minute; The solution obtained is called the reduced sample solution. c) Alkylation of the reduced sulfhydryl (- SH) groups (9), present in the cistern residues. This step is carried out by adding the alkylation solution in a 10: 1 volume ratio (reduced sample solution: alkylation solution). The time for alkylation is preferably 5 minutes at room temperature. d) Separation of the proteins in a sodium polyacrylamide-dodecyl sulfate gel (SDS-PAGE) (10). The separation was carried out according to the standard protocol described by Sambrook 2001, applying an electric current of 150 V for 45 minutes. In this case the separation of the proteins was carried out in 10 and 12.5% polyacrylamide gels (although the concentration of polyacrylamide used in the gels depends on the molecular weight of the proteins of interest to be separated). e) Cut the protein bands of the SDS-PAGE gel (11). This is done by cutting the region where the band that corresponds to the protein of interest in the gel is located. Subsequently the bands with the protein of interest are cut into smaller fragments (lmm x lmm). f) Remove excess water present in the gel fragments in addition to fixing the proteins (12), this is carried out by fixing and dehydrating the gel fragments. This is done by the direct addition of acetonitrile to the gel fragments until they are covered and incubating for 5 minutes at room temperature, then the excess acetonitrile is removed from the samples, and these are dried by vacuum centrifugation for 10 minutes. This stage is recommended to reduce the effects of acetonitrile on trypsin when digestion is performed. g) Tryptic digestion of the proteins present in the fixed and dehydrated gel fragments (13). This is done by adding a trypsin solution (V511A Promega, 10
Figure imgf000009_0001
in NH 4 HCO 3 , 25 mM) sufficient to cover the previously fixed and dehydrated gel fragments. Subsequently, the gel fragments are incubated at a temperature of 60 ° C for 30 minutes, at this stage the trypsin hydrolysis reaction near residues modified with Uniblue A is inhibited, therefore the hydrolysis reaction is carried out mostly in arginine residues obtaining larger proteolytic fragments, achieving an improvement in the coverage of peptide sequencing, it should be noted that at this stage the ions of the N-terminal group preceded by a plant have an additional mass corresponding to Uniblue A (484.0 Da), which facilitates its detection by mass spectrometry, and each lysine modification affects the mass of amino acid fragments in the N or C terminal direction. h) Extraction of the peptides from the gel fragments (14). This is accomplished by the addition of a 0.1% (v / v) acetonitrile / trifluoroacetic acid solution in a 1: 1 ratio to the gel fragments followed by incubation at 60 ° C for 15 minutes. In order to obtain a greater coverage of the peptide sequence it is recommended to use long extraction times. After extraction it is important to transfer the extraction solution to a new tube. i) Dry the peptides (15), this is carried out by removing the excess acetonitrile / trifluoroacetic acid solution by vacuum centrifugation until the dried peptides are obtained. Preparation and analysis of the peptides extracted by mass spectrometry (16). The extracted peptides are recovered by the addition of approximately 20 μΐ. of a 0.1% (v / v) formic acid solution, the solution with the peptides is transferred in polypropylene vials (FAMOS) to nano LC-MS MS. Peptide analysis can be performed, e.g. ex. in a nano LC-ESI-ion trap. The resulting spectrum can be evaluated using automatic investigation algorithms such as the X! Tandem or OMSSA. To quantify the number of covalent modifications, mass changes of + 484.03989 Da (monoisotopic) for Uniblue A are taken into account.
Otro objeto de la presente invención se refiere a un Kit para visualizar instantáneamente proteínas para su posterior caracterización usando espectrometría de masas. El pregonado Kit comprende los siguientes reactivos, almacenados de manera independiente: Another object of the present invention relates to a Kit for instantly visualizing proteins for subsequent characterization using mass spectrometry. The proclaimed Kit comprises the following reagents, stored independently:
a) Una solución amortiguadora A. Esta solución tiene un pH en el rango de 8 a 9, preferentemente consiste en una solución de carbonato de sodio a una concentración 100 mM y una solución de dodecilsulfato sódico (SDS) a una concentración de 10% (p/v).  a) A buffer solution A. This solution has a pH in the range of 8 to 9, preferably it consists of a solution of sodium carbonate at a concentration of 100 mM and a solution of sodium dodecyl sulfate (SDS) at a concentration of 10% ( p / v).
b) Solución de colorante Uniblue A. El colorante Umblue A es disuelto en solución amortiguadora A, a una concentración de 60 mM.  b) Uniblue A dye solution. The Umblue A dye is dissolved in buffer solution A, at a concentration of 60 mM.
c) Solución reductora. Esta solución tiene un pH 6.8, y consiste en una solución al 20% (v/v) de glicerol, Tris HC1 200 mM y 20 mM ditiotreitol (DTT).  c) Reducing solution. This solution has a pH 6.8, and consists of a 20% (v / v) solution of glycerol, 200 mM Tris HC1 and 20 mM dithiothreitol (DTT).
d) Solución de alquilación: Consiste en una solución de iodoacetamida (IAA) a una concentración de 550 mM en solución amortiguadora A.  d) Alkylation solution: It consists of a solution of iodoacetamide (IAA) at a concentration of 550 mM in buffer solution A.
e) Control positivo: 3 mg de albúmina de suero bovino (ASB) liofilizada, para reconstituir en 1 mL de solución amortiguadora A.  e) Positive control: 3 mg of lyophilized bovine serum albumin (ASB), to reconstitute in 1 mL of buffer solution A.
Las instrucciones para usar el Kit se describen a continuación: The instructions for using the Kit are described below:
a) Adicionar 10 de solución colorante a 90 μL· de solución muestra que contiene la proteína de interés, para obtener una solución muestra coloreada,  a) Add 10 dye solution to 90 μL · of sample solution containing the protein of interest, to obtain a colored sample solution,
b) Calentar la solución muestra coloreada a 100 °C por 1 minuto,  b) Heat the colored sample solution at 100 ° C for 1 minute,
c) Agregar 100 de la solución reductora, a la solución muestra coloreada caliente, manteniendo el calentamiento a 100°C por 1 minuto,  c) Add 100 of the reducing solution to the hot colored sample solution, keeping the heating at 100 ° C for 1 minute,
d) Agregar 20 iL de la solución de alquilación a cada solución muestra coloreada caliente de c),  d) Add 20 iL of the alkylation solution to each hot colored sample solution of c),
e) Incubar la solución muestra coloreada de d) a temperatura ambiente por 5 minutos. f) Cargar la solución muestras coloreada en el gel de SDS-PAGE, e) Incubate the colored sample solution of d) at room temperature for 5 minutes. f) Load the colored sample solution into the SDS-PAGE gel,
g) Separar las proteínas de la muestra coloreada de f) mediante electroforesis (por ejemplo 150 V por 45 minutos, Bio-Rad Protean3 Minigel 10% Acrilamida). Cada uno de los ejemplos posteriores hacen referencia a la visualización de las bandas de proteínas en un gel de SDS-PAGE, comparando la tinciones de Uniblue A y azul de Coomassie, esto para mostrar la visualización instantánea de las proteínas para su posterior caracterización mediante espectrometría de MS MS. Ejemplo 1. Modificación covalente de proteínas para su visualización instantánea empleando el Kit motivo de esta invención en una muestra de albúmina de suero bovino (ASB) y posterior caracterización mediante espectrometría de masas.  g) Separate the proteins from the colored sample of f) by electrophoresis (for example 150 V for 45 minutes, Bio-Rad Protean3 Minigel 10% Acrylamide). Each of the subsequent examples refers to the visualization of the protein bands in an SDS-PAGE gel, comparing the Uniblue A and Coomassie blue stains, this to show the instant visualization of the proteins for subsequent characterization by spectrometry of MS MS. Example 1. Covalent modification of proteins for instant visualization using the reason kit of this invention in a sample of bovine serum albumin (ASB) and subsequent characterization by mass spectrometry.
I. Preparación de la muestra. I. Preparation of the sample.
Reconstituir 3.0 mg de ASB liofilizada en 1 mL de solución amortiguadora A en un tubo Eppendorf.  Reconstitute 3.0 mg of lyophilized ASB in 1 mL of buffer solution A in an Eppendorf tube.
Π. Método de empleo del Kit. Π Method of use of the Kit.
Agregar 10 ÍL de solución colorante (Uniblue A) a 90 iL* de la solución de ASB (relación de volúmenes 1:9). Posteriormente las muestras fueron incubadas a 100 °C por 1 minuto. Enseguida se agregaron 100 μΐ, de la solución reductora (relación de volúmenes 1:1) a la mezcla mencionada anteriormente y se incubaron a 100 °C por 1 minuto. Finalmente, se adicionaron 20 μΐ, de la solución de alquilación e incubar por 5 minutos a temperatura ambiente. Add 10 µl of dye solution (Uniblue A) to 90 µl * of the ASB solution (ratio of volumes 1: 9). Subsequently the samples were incubated at 100 ° C for 1 minute. Then 100 μΐ of the reducing solution (ratio of 1: 1 volumes) was added to the above-mentioned mixture and incubated at 100 ° C for 1 minute. Finally, 20 μΐ of the alkylation solution was added and incubated for 5 minutes at room temperature.
ΙΠ. Separación de las proteínas en un gel de SDS-PAGE. ΙΠ Separation of proteins in an SDS-PAGE gel.
Para la separación de las muestras se emplearon geles de una concentración de acrilamida de 12.5% (0.75 mm de espesor). Una vez listas las muestras, se cargaron volúmenes de 2 μΐ de la muestra de ASB en cada uno de los carriles del gel. Adicionalmente se empleó un marcador molecular Kaleidoscope™ de Bio-Rad (en este caso se empleó un volumen de 5 μί). La separación de las proteínas se llevó a cabo a un voltaje de 150 V por 45 minutos (ver figura 2-a y 2-b).  For the separation of the samples, gels with an acrylamide concentration of 12.5% (0.75 mm thick) were used. Once the samples were ready, 2 μ 2 volumes of the ASB sample were loaded into each of the gel rails. Additionally, a Bio-Rad Kaleidoscope ™ molecular marker was used (in this case a volume of 5 μί was used). The separation of the proteins was carried out at a voltage of 150 V for 45 minutes (see Figure 2-a and 2-b).
IV. Preparación de la muestra para caracterización. Primero se llevó a cabo el corte de las bandas correspondientes a las proteínas de interés del gel de SDS-PAGE. Posteriormente las bandas con la proteína de interés se cortaron en fragmentos más pequeños (aproximadamente lmm x lmm). Después, se adicionó acetonitrilo a los fragmentos de gel con la finalidad de fijar y deshidratar las proteínas presentes en los fragmentos de gel. Finalmente el exceso de acetonitrilo fue removido de las muestras, y posteriormente estas fueron secadas mediante centrifugación al vacio por 10 minutos. Posteriormente, se realizó la digestión tríptica de las proteínas presentes en los fragmentos de gel. Esto se realizó mediante la adición de una solución de tripsina (V511A Promega, 10 ng/μL en NH4HCO3, 25 mM), suficiente para cubrir los fragmentos de gel previamente fijados y deshidratados. Posteriormente, los fragmentos de gel fueron incubados a una temperatura de 60°C durante 30 minutos. Después de la digestión, el siguiente paso fue la extracción de los péptídos de los fragmentos de gel. Esto se realizó por la adición de una solución de acetonitrilo/ácido trifluoroacético 0.1% (v/v) en relación 1: 1 a los fragmentos de gel seguido de la incubación a 60 °C por 15 minutos. Después de la extracción, la solución fue transferida a un tubo nuevo.IV. Sample preparation for characterization. First, the bands corresponding to the proteins of interest of the SDS-PAGE gel were cut. Subsequently the bands with the protein of interest were cut into smaller fragments (approximately lmm x lmm). Then, acetonitrile was added to the gel fragments in order to fix and dehydrate the proteins present in the gel fragments. Finally, the excess acetonitrile was removed from the samples, and subsequently these were dried by vacuum centrifugation for 10 minutes. Subsequently, tryptic digestion of the proteins present in the gel fragments was performed. This was done by adding a trypsin solution (V511A Promega, 10 ng / μL in NH 4 HCO 3 , 25 mM), sufficient to cover the previously fixed and dehydrated gel fragments. Subsequently, the gel fragments were incubated at a temperature of 60 ° C for 30 minutes. After digestion, the next step was the extraction of the peptides from the gel fragments. This was accomplished by the addition of a 0.1% (v / v) acetonitrile / trifluoroacetic acid solution in 1: 1 ratio to the gel fragments followed by incubation at 60 ° C for 15 minutes. After extraction, the solution was transferred to a new tube.
Finalmente, el exceso de líquido fue eliminado por centrifugación a vacío hasta obtener los péptídos secos. Finally, the excess liquid was removed by vacuum centrifugation until the dried peptides were obtained.
Caracterización de la muestra mediante espectrometría de masas. Sample characterization by mass spectrometry.
Los péptídos extraídos fueron recuperados por la adición de aproximadamente 20 μΐ, de una solución de ácido fórmico 0.1% (v/v), la solución con los péptídos fue transferida en viales de polipropileno (FAMOS) para nano LC-MS/MS. El análisis de los péptídos se llevó a cabo usando un nano LC-ESI- trampa de iones. El espectro resultante fue evaluado por el uso de algoritmos de investigación automática X!Tandem y OMSSA. Para cuantificar el número de modificaciones covalentes, se tomó en cuenta el cambio en masa de + 484.03989 Da (monoisotópico) en los aminoácidos modificados por Uniblue A, (ver figura 5-a, 5-b y 5-c).  The extracted peptides were recovered by the addition of approximately 20 μΐ, of a 0.1% (v / v) formic acid solution, the solution with the peptides was transferred in polypropylene vials (FAMOS) to nano LC-MS / MS. Peptide analysis was carried out using a nano LC-ESI-ion trap. The resulting spectrum was evaluated by the use of X! Tandem and OMSSA automatic research algorithms. To quantify the number of covalent modifications, the mass change of + 484.03989 Da (monoisotopic) in the amino acids modified by Uniblue A, was taken into account (see Figure 5-a, 5-b and 5-c).
Ejemplo 2. Modificación covalente de proteínas para su visualización instantánea empleando el Kit motivo de esta invención en una muestra de un anticuerpo recombinante (Rituximab). Example 2. Covalent modification of proteins for instant visualization using the Motif Kit of this invention in a sample of a recombinant antibody (Rituximab).
I. Preparación de la muestra.  I. Preparation of the sample.
Para este procedimiento se llevó a cabo un intercambio de una solución amortiguadora no compatible por solución amortiguadora A. Primero, 250 iL de una solución de Rituximab recombinante (500mg 50mL) fueron colocados en un tubo para ultrafiltración Amicon® Ultra (Millipore™) con capacidad de 0.5 mL y una membrana de 3000 MWCO (Molecular Weight Cut Off, [Da]). Posteriormente, se agregaron 250 μΐ- de solución amortiguadora A. For this procedure, an exchange of a non-compatible buffer solution was carried out with buffer solution A. First, 250 iL of a recombinant Rituximab solution (500mg 50mL) were placed in a Amicon® Ultra ultrafiltration tube (Millipore ™) with a capacity of 0.5 mL and a 3000 MWCO membrane (Molecular Weight Cut Off, [Da]). Subsequently, 250 μΐ- of buffer solution A.
Después la muestra fue centrifugada a 14,000 x g por 30 minutos en una centrifuga para tubos Eppendorf. Enseguida se adicionó un volumen suficiente de solución amortiguadora A para tener un alcanzar un volumen de 0.5 mL (máxima capacidad del tubo Eppendorf). Repetir este procedimiento al menos cinco veces. Después de repetir el procedimiento anterior por última vez, el Rituximab fue resuspendido en 125 )ÍL de solución amortiguadora A, la concentración final de esta solución fue de 20 mg/mL. The sample was then centrifuged at 14,000 x g for 30 minutes in a centrifuge for Eppendorf tubes. A sufficient volume of buffer solution A was then added to have a volume of 0.5 mL (maximum capacity of the Eppendorf tube). Repeat this procedure at least five times. After repeating the previous procedure for the last time, Rituximab was resuspended in 125) I of buffer solution A, the final concentration of this solution was 20 mg / mL.
Π. Método de empleo del Kit. Π Method of use of the Kit.
Se agregaron 10 γΛ-, de solución colorante (Uniblue A) a 90 de la solución de Rituximab (relación de volúmenes 1:9). Posteriormente las muestras fueron incubadas a 100 °C por 1 minuto. Enseguida se agregaron 100 de la solución reductora (relación de volúmenes 1: 1) a la mezcla mencionada anteriormente y se incubaron a 100 °C por 1 minuto. Finalmente, se adicionaron 20 iL de la solución de alquilación e incubar 5 minutos a temperatura ambiente.  10 γΛ-, dye solution (Uniblue A) was added to 90 of the Rituximab solution (ratio of volumes 1: 9). Subsequently the samples were incubated at 100 ° C for 1 minute. Then 100 of the reducing solution (1: 1 volume ratio) was added to the above-mentioned mixture and incubated at 100 ° C for 1 minute. Finally, 20 iL of the alkylation solution was added and incubated 5 minutes at room temperature.
III. Separación de las proteínas en un gel de SDS-PAGE. III. Separation of proteins in an SDS-PAGE gel.
Para la separación de las muestras se emplearon geles de una concentración de acrilamida de 12.5% (0.75 mm de espesor). Una vez listas las muestras, se cargaron volúmenes de 2 μΐ- de la muestra de Rituximab en cada uno de los carriles del gel. Se empleó un marcador molecular Kaleidoscope™ de BioRad (en este caso se empleó un volumen de 5 μΐ . La separación de las proteínas se llevó a cabo a un voltaje de 150 V por 45 minutos (ver figura 3-a y 3-b).  For the separation of the samples, gels with an acrylamide concentration of 12.5% (0.75 mm thick) were used. Once the samples were ready, 2 μ 2- volumes of the Rituximab sample were loaded into each of the gel rails. A BioRad Kaleidoscope ™ molecular marker was used (in this case a volume of 5 μΐ was used. The separation of the proteins was carried out at a voltage of 150 V for 45 minutes (see Figure 3-a and 3-b).
Ejemplo 3. Modificación covalente de proteínas para su visualización instantánea empleando el Kit, este caso, en una muestra de Escherichia coli, cepa TOP10, con y sin expresión de una proteína recombinante con el plásmido pMAL-c4x, que codifica para una proteína de fusión. Example 3. Covalent modification of proteins for instant visualization using the Kit, this case, in a sample of Escherichia coli, strain TOP10, with and without expression of a recombinant protein with plasmid pMAL-c4x, which codes for a fusion protein .
La fusión es dada por una proteína de unión a la maltosa y el gen lacZa. En este ejemplo se realizó un control de expresión de una proteína.  Fusion is given by a maltose binding protein and the lacZa gene. In this example, a protein expression control was performed.
I. Preparación de la muestra. La propagación de las cepas de E. coli fue llevada a cabo en 50 mL del medio TB Overnight Express (Novagen Inc.). Adicionalmente, para la cepa recombinante se agregaron 150 uL del antibiótico carbencilina al medio TB para evitar el crecimiento de otros microorganismos. Después el medio de cultivo se inoculó con 100 uL de una solución de E. coli. El cultivo de E. coli fue llevado a cabo por 16 horas a 37°C y una agitación de 250 rpm. Después de la incubación, el medio de cultivo con células fue separado en volúmenes de 7mL en tubos cónicos de 15 mL y centrifugado a 5,000 x g a una temperatura de 4°C por 15 minutos. Enseguida el sobrenadante fue removido y las células de E. coli precipitadas, en el fondo del tubo, fueron resuspendidas con la adición de 4.5 mL de solución amortiguadora A. Posteriormente, se llevó a cabo el rompimiento de la pared celular de E. coli para obtener las proteínas presentes en el interior de las células, esto fue realizado por ultrasonicación durante 15 minutos. Después, la muestra fue centrifugada a 10,000 x g por 15 minutos a 4°C, y el sobrenadante fue recuperado. I. Preparation of the sample. The propagation of E. coli strains was carried out in 50 mL of TB Overnight Express medium (Novagen Inc.). Additionally, 150 uL of the antibiotic carbencillin was added to the TB medium for the recombinant strain to prevent the growth of other microorganisms. The culture medium was then inoculated with 100 uL of a solution of E. coli. The culture of E. coli was carried out for 16 hours at 37 ° C and stirring of 250 rpm. After incubation, the cell culture medium was separated into 7mL volumes in 15 mL conical tubes and centrifuged at 5,000 xg at a temperature of 4 ° C for 15 minutes. Then the supernatant was removed and the precipitated E. coli cells, at the bottom of the tube, were resuspended with the addition of 4.5 mL of buffer solution A. Subsequently, the E. coli cell wall rupture was performed to To obtain the proteins present inside the cells, this was done by ultrasonication for 15 minutes. Then, the sample was centrifuged at 10,000 xg for 15 minutes at 4 ° C, and the supernatant was recovered.
Las proteínas presentes en el sobrenadante fueron precipitadas por la adición de 0.5 mL de una solución de TCA/acetona (lg TCA/mL de acetona) en relación de volúmenes 9:1 (Sobrenadante: solución TCA/acetona). Este procedimiento se realizó a una temperatura de 4°C por 1-2 horas. Después de la precipitación, las muestras fueron centrifugadas a 10,000 x g por 15 minutos a 4°C. El sobrenadante fue desechado y las proteínas precipitadas fueron lavadas con acetona (90%) al menos tres veces (centrifugar a 10,000 x g por 15 minutos entre cada lavada). Finalmente el exceso de acetona fue eliminado y las proteínas fueron reconstituidas en 100 uL de solución amortiguadora A.  The proteins present in the supernatant were precipitated by the addition of 0.5 mL of a TCA / acetone solution (lg TCA / mL of acetone) in a ratio of 9: 1 volumes (Supernatant: TCA / acetone solution). This procedure was performed at a temperature of 4 ° C for 1-2 hours. After precipitation, the samples were centrifuged at 10,000 x g for 15 minutes at 4 ° C. The supernatant was discarded and the precipitated proteins were washed with acetone (90%) at least three times (centrifuge at 10,000 x g for 15 minutes between each wash). Finally, the excess acetone was removed and the proteins were reconstituted in 100 uL of buffer solution A.
Método de empleo del Kit. Method of use of the Kit.
Se agregaron 10 de la solución colorante (Uniblue A) a 90 μL de la solución de E. coli (relación de volúmenes 1:9). Las muestras fueron incubadas a 100 °C por 1 minuto. Se adicionaron 100
Figure imgf000014_0001
de la solución reductora (relación de volúmenes 1:1) a las muestras y después fueron incubadas a 100 °C por 1 minuto. Finalmente, se adicionaron 20 μί de la solución de alquilación a las muestras y se incubaron por 5 minutos a temperatura ambiente. 10 of the dye solution (Uniblue A) was added to 90 μL of the E. coli solution (ratio of volumes 1: 9). The samples were incubated at 100 ° C for 1 minute. 100 were added
Figure imgf000014_0001
of the reducing solution (ratio of volumes 1: 1) to the samples and then incubated at 100 ° C for 1 minute. Finally, 20 μί of the alkylation solution was added to the samples and incubated for 5 minutes at room temperature.
III. Separación de las proteínas en un gel de SDS-PAGE. Para la separación de las muestras se emplearon geles de una concentración de acnlamida de 12.5% (0.75 mm de espesor). Una vez listas las muestras, se cargaron en volúmenes de 5 iL de la muestra de ASB en cada uno de los carriles del gel. Se empleó un marcador molecular Kaleidoscope™ de BioRad (en este caso se empleó un volumen de 5 μί). La separación de las proteínas se llevó a cabo a un voltaje de 150 V por 45 minutos (4-a, 4-b y 4-c). III. Separation of proteins in an SDS-PAGE gel. For the separation of the samples, gels of an acnlamide concentration of 12.5% (0.75 mm thick) were used. Once the samples were ready, they were loaded in volumes of 5 iL of the ASB sample in each of the gel rails. A BioRad Kaleidoscope ™ molecular marker was used (in this case a volume of 5 μί was used). The separation of the proteins was carried out at a voltage of 150 V for 45 minutes (4-a, 4-b and 4-c).
Tabla 1: Análisis de péptído de ASB (carga 2+) identificado mediante OMSSA (tolerancia 2 Da, 0.8 Da para fragmentos) Table 1: ASB peptide analysis (load 2+) identified by OMSSA (tolerance 2 Da, 0.8 Da for fragments)
Secuencia del péptido de ASB  ASB peptide sequence
Figure imgf000015_0001
Figure imgf000015_0001
Sec ID No.1 Sec ID No.1
K V P Q V S T P T L V E V S R  K V P Q V S T P T L V E V S R
Lys Val Pro Glm Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Lys Val Pro Glm Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg
Tabla 2: Fragmentos N-terminales del péptido sin y con modificación con Uniblue A.Table 2: N-terminal fragments of the peptide without and with modification with Uniblue A.
Sec. Código Frag. sin modificación con Uniblue A Sec. Code Frag. without modification with Uniblue A
AA AA N-term Teórico Medido teórico Medido  AA AA N-term Theoretical Measured Theoretical Measured
K(l) i Lys bl 129.10 - 613.14 613.19  K (l) i Lys bl 129.10 - 613.14 613.19
V Val b2 228.17 228.07 712.21 712.37  V Val b2 228.17 228.07 712.21 712.37
P Pro b3 325.22 325.15 809.26 - P Pro b3 325.22 325.15 809.26 -
Q Glm 453.28 453.32 937.32 937.49 Q Glm 453.28 453.32 937.32 937.49
V Val b5 552.35 552.43 1036.39 1036.53  V Val b5 552.35 552.43 1036.39 1036.53
s Ser b6 639.38 639.52 1123.42 1123.59  s Ser b6 639.38 639.52 1123.42 1123.59
T Tin- b7 740.43 740.68 1224.47 1224.71  T Tin-b7 740.43 740.68 1224.47 1224.71
P Pro b8 837.48 - 1321.52 - P Pro b8 837.48 - 1321.52 -
T Tin- b9 938.53 - 1422.57 -T Tin-b9 938.53 - 1422.57 -
L Leu blO 1051.62 1051.88 1535.66 1535.66 L Leu blO 1051.62 1051.88 1535.66 1535.66
V Val bll 1150.68 (1150.7)* 1634.72 1634.81  V Val bll 1150.68 (1150.7) * 1634.72 1634.81
E Glu bl2 1279.73 1279.94 1763.77 1763.89  E Glu bl2 1279.73 1279.94 1763.77 1763.89
V Val bl3 1378.79 1378.89 1862.83 1862.93  V Val bl3 1378.79 1378.89 1862.83 1862.93
s Ser bl4 1465.83 1465.73 1949.87 1950.63**  s Ser bl4 1465.83 1465.73 1949.87 1950.63 **
R Arg bl5 - - - -  R Arg bl5 - - - -
* Identificado solamente mediante OMSSA para la tolerancia más alta * Identified only by OMSSA for the highest tolerance
** Análisis manual mediante mmass, error 0.76 Da ** Manual analysis by mmass, error 0.76 Da
Letra Estilo normal: análisis manual con mmass (tolerancia 0.5 Da),  Normal style letter: manual analysis with mmass (tolerance 0.5 Da),
Letra Estilo negrita análisis automática con OMSSA (tolerancia 0.8 Da para fragmentos, modificación opcional con Uniblue A con 484.0 Da). Bold style letter automatic analysis with OMSSA (tolerance 0.8 Da for fragments, optional modification with Uniblue A with 484.0 Da).
Tabla 3: Fragmentos C-terminales del péptido sin y con modifícación con Uniblue A Table 3: C-terminal fragments of the peptide without and with modification with Uniblue A
Sec. Código Frag. sin modifícación con Uniblue A Sec. Code Frag. without modification with Uniblue A
AA C- AA C-
AA term Teórico medido teórico medido AA term Theoretical measured theoretical measured
K(l) Lys yl5 - - - - K (l) Lys yl5 - - - -
V Val yl4 1511.84 1512.32 1511.84 -V Val yl4 1511.84 1512.32 1511.84 -
P Pro yl3 1412.77 1412.98 1412.77 1413.04 P Pro yl3 1412.77 1412.98 1412.77 1413.04
Q Glm yl2 1315.72 1316.04 1315.72 1315.78  Q Glm yl2 1315.72 1316.04 1315.72 1315.78
V Val yl l 1187.66 1187.89 1187.66 1187.94 6 s Ser ylO 1088.60 1088.79 1088.60 1088.79 V Val yl l 1187.66 1187.89 1187.66 1187.94 6 s Ser ylO 1088.60 1088.79 1088.60 1088.79
T Tin- y9 1001.56 1001.80 1001.56 1001.85  T Tin- y9 1001.56 1001.80 1001.56 1001.85
P Pro y8 900.52 900.74 900.52 900.71  P Pro y8 900.52 900.74 900.52 900.71
T Tin- y7 803.46 803.54 803.46 803.71  T Tin- y7 803.46 803.54 803.46 803.71
L Leu y6 702.42 702.42 702.42 702.35  L Leu y6 702.42 702.42 702.42 702.35
V Val y5 589.33 589.55 589.33 589.53  V Val y5 589.33 589.55 589.33 589.53
E Glu y4 490.26 490.30 490.26 490.44  E Glu y4 490.26 490.30 490.26 490.44
V Val y3 361.22 361.27 361.22 361.23  V Val y3 361.22 361.27 361.22 361.23
S Ser y2 262.15 262.06 262.15 - S Ser y2 262.15 262.06 262.15 -
R Arg yi 175.12 - 175.12 - Estos resultados indican, que una modificación de peso molecular de 484.0 Da está presente en la posición 1 del péptido, Usina (lys, K). R Arg yi 175.12-175.12 - These results indicate that a molecular weight modification of 484.0 Da is present at position 1 of the peptide, Usina (lys, K).

Claims

REIVINDICACIONES
1. Un método para modificación covalente de proteínas para su visualización instantánea y posterior caracterización usando espectrometría de masas, caracterizado porque comprende las etapas de: 1. A method for covalent modification of proteins for instant visualization and subsequent characterization using mass spectrometry, characterized in that it comprises the steps of:
a) Teñir una muestra que contiene la proteína de interés en solución, con una solución colorante con un grupo funcional, para reaccionar en una adición nucleofilica con los grupos amino de la proteína, preferentemente en los aminoácidos lisina de la secuencia peptidíca; y opcionalmente tiene al menos otro grupo funcional ionizable para dar mayor solubilidad a la tinción en agua, posteriormente dar mayor solubilidad a los péptidos e influye su ionización por espectrometría de masas;  a) Staining a sample containing the protein of interest in solution, with a dye solution with a functional group, to react in a nucleophilic addition with the amino groups of the protein, preferably in the lysine amino acids of the peptide sequence; and optionally it has at least one other ionizable functional group to give greater solubility to water staining, subsequently give greater solubility to the peptides and influence their ionization by mass spectrometry;
b) Reducir los enlaces disulfuro de la proteína contenida en la solución muestra obtenida en la etapa a), particularmente intercambiando un grupo sulfidrilo (-SH) presente en el enlace disulfuro por ditiotreitol; llevando esto a cabo mediante la adición de una solución reductora, preferentemente en una relación de volúmenes 1:1 (solución muestra : solución reductora), y calentando por incubación a 100°C durante un tiempo de al menos 1 minuto, la solución obtenida es denominada solución muestra reducida;  b) Reduce the disulfide bonds of the protein contained in the sample solution obtained in step a), particularly by exchanging a sulfhydryl group (-SH) present in the disulfide bond for dithiothreitol; carrying out this by adding a reducing solution, preferably in a ratio of 1: 1 volumes (sample solution: reducing solution), and heating by incubation at 100 ° C for a time of at least 1 minute, the solution obtained is called a reduced sample solution;
c) Alquilar los grupos sulfhidrilo (— SH) de la solución muestra reducida, presentes en los residuos de cisterna; esto se lleva a cabo agregando una solución de alquilación en una relación de volúmenes preferentemente 10:1 (solución muestra reducida : solución de alquilación) y mantener a temperatura ambiente durante al menos 5 minutos; aunque el tiempo de incubación puede ser prolongado arriba de 5 minutos sin afectar la reacción; la solución obtenida es denominada solución de proteína alquilada;  c) Rent the sulfhydryl groups (- SH) of the reduced sample solution, present in the cistern residues; this is accomplished by adding an alkylation solution in a preferably 10: 1 volume ratio (reduced sample solution: alkylation solution) and maintaining at room temperature for at least 5 minutes; although the incubation time can be prolonged above 5 minutes without affecting the reaction; the solution obtained is called an alkylated protein solution;
d) Separar las proteínas contenidas en la solución de proteína alquilada, en un gel de poliacrilamida-dodecil sulfato de sodio (SDS-PAGE), de acuerdo al protocolo estándar descrito por Sambrook, aplicando una corriente eléctrica preferentemente de 150 V por 45 minutos; es posible disminuir el voltaje e incrementar el tiempo de separación, así como incrementar el voltaje y disminuir el tiempo de separación; e) Cortar la bandas de interés del gel de SDS-PAGE, en fragmentos de lmm x lmm; f) Eliminar el exceso de agua presente en los fragmentos de gel además de fijar las proteínas, esto se lleva a cabo por la adición directa de acetonitrilo a los fragmentos de gel incuban por 5 minutos a temperatura ambiente, posteriormente el exceso de acetonitrilo es removido de las muestras, y posteriormente estas son secadas mediante centrifugación al vacio por 10 minutos; d) Separate the proteins contained in the alkylated protein solution, in a sodium polyacrylamide dodecyl sulfate gel (SDS-PAGE), according to the standard protocol described by Sambrook, applying an electric current preferably of 150 V for 45 minutes; it is possible to decrease the voltage and increase the separation time, as well as increase the voltage and decrease the separation time; e) Cut the bands of interest of the SDS-PAGE gel into fragments of lmm x lmm; f) Remove the excess water present in the gel fragments in addition to fixing the proteins, this is carried out by the direct addition of acetonitrile to the gel fragments incubated for 5 minutes at room temperature, then the excess of Acetonitrile is removed from the samples, and subsequently these are dried by vacuum centrifugation for 10 minutes;
g) Digestión tríptica de las proteínas presentes en los fragmentos de gel fijados y deshidratados, esto se realiza cubriendo los fragmentos de gel, con una solución de tripsina (V511A Promega, 10
Figure imgf000019_0001
en NH4HCO3, 25 mM), e incubar a una temperatura de 60°C durante 30 minutos; g) Triptych digestion of the proteins present in the fixed and dehydrated gel fragments, this is done by covering the gel fragments, with a trypsin solution (V511A Promega, 10
Figure imgf000019_0001
in NH 4 HCO 3 , 25 mM), and incubate at a temperature of 60 ° C for 30 minutes;
h) Extraer los péptidos de los fragmentos de gel digeridos en g), esto se lleva a cabo adicionando una solución de acetonitrilo/ácido trifluoroacético 0.1% (v/v) en relación 1:1 a los fragmentos de gel digeridos, e incubar a 60 °C por 15 minutos; i) Secar los péptidos, eliminando el exceso de solución de acetonitrilo/ácido trifluoroacético por centrifugación a vacío hasta obtener los péptidos secos;  h) Extract the peptides from the digested gel fragments in g), this is accomplished by adding a 0.1% (v / v) acetonitrile / trifluoroacetic acid solution in 1: 1 ratio to the digested gel fragments, and incubate at 60 ° C for 15 minutes; i) Dry the peptides, removing excess acetonitrile / trifluoroacetic acid solution by vacuum centrifugation until the dry peptides are obtained;
j) Preparar los péptidos extraídos para análisis mediante espectrometría de masas; los péptidos secos, son recuperados por la adición de aproximadamente 20 uL de una solución de ácido fórmico 0.1% (v/v), la solución con los péptidos es transferida en viales de polipropileno (FAMOS) para nano LC-MS MS.  j) Prepare the extracted peptides for analysis by mass spectrometry; The dried peptides are recovered by the addition of approximately 20 uL of a 0.1% (v / v) formic acid solution, the solution with the peptides is transferred in polypropylene vials (FAMOS) to nano LC-MS MS.
2. El método para modificación covalente de proteínas de conformidad con la reivindicación 1, caracterizado porque en la etapa a), el colorante preferentemente es Uniblue A, a una concentración de 60 mM, disuelto en una solución de carbonato de sodio (100 mM) y dodecilsulfato sódico (SDS) al 10% (p/v) en solución con agua bidestilada, a un pH en el rango de 8 a 9. 2. The method for covalent modification of proteins according to claim 1, characterized in that in step a), the dye is preferably Uniblue A, at a concentration of 60 mM, dissolved in a solution of sodium carbonate (100 mM) and 10% (w / v) sodium dodecyl sulfate (SDS) in solution with double distilled water, at a pH in the range of 8 to 9.
3. El método para modificación covalente de proteínas de conformidad con la reivindicaciones 1 y 2, caracterizado porque en la etapa a), la tinción se lleva a cabo preferentemente calentando a 100°C durante 1 minuto; aunque la temperatura de calentamiento puede ser menor a 100°C, con tiempos de reacción prolongados. 3. The method for covalent modification of proteins according to claims 1 and 2, characterized in that in step a), staining is preferably carried out by heating at 100 ° C for 1 minute; although the heating temperature may be less than 100 ° C, with prolonged reaction times.
4. El método para modificación covalente de proteínas de conformidad con la reivindicación 1, caracterizado porque en la etapa a), la muestra que contiene la proteína de interés, opcionalmente consiste en un: 4. The method for covalent modification of proteins according to claim 1, characterized in that in step a), the sample containing the protein of interest, optionally consists of:
a) Una muestra con proteína seca (liofilizada);  a) A sample with dried protein (lyophilized);
b) Una muestra de proteínas en solución en una concentración suficiente y una solución amortiguadora compatible; c) Una muestra de proteína en solución en una baja concentración y solución amortiguadora no compatible, (dígase de solución amortiguadora no compatible como aquella que contiene grupos amino, p.ej.: Tris). b) A sample of proteins in solution in a sufficient concentration and a compatible buffer solution; c) A sample of protein in solution in a low concentration and non-compatible buffer solution, (say non-compatible buffer solution such as that containing amino groups, eg: Tris).
5. El método para modificación covalente de proteínas de conformidad con la reivindicación 4, caracterizado porque las muestras de proteína seca, se disuelve la muestra en una solución de carbonato de sodio (100 mM) y dodecilsulfato sódico (SDS) al 10% (p/v) en solución con agua bidestilada, a un pH en el rango de 8 a 9. 5. The method for covalent modification of proteins according to claim 4, characterized in that the dry protein samples are dissolved in a solution of sodium carbonate (100 mM) and 10% sodium dodecyl sulfate (SDS) (p / v) in solution with double-distilled water, at a pH in the range of 8 to 9.
6. El método para modificación covalente de proteínas de conformidad con la reivindicación 4, caracterizado porque las muestras de proteínas en solución en una concentración suficiente y una solución amortiguadora compatible; son utilizadas tal cual, en el método propuesto. 6. The method for covalent modification of proteins according to claim 4, characterized in that the protein samples in solution in a sufficient concentration and a compatible buffer solution; They are used as is, in the proposed method.
7. El método para modificación covalente de proteínas de conformidad con la reivindicación 4, caracterizado porque las muestra de proteína en solución en una baja concentración y solución amortiguadora no compatible; opcionalmente son: 7. The method for covalent modification of proteins according to claim 4, characterized in that the protein samples in solution in a low concentration and non-compatible buffer solution; optionally they are:
a) precipitadas usando TC A/acetona [lg TCA/mL acetona, previamente enfriada a 4 °C] y posteriormente resuspendidas en una solución amortiguadora [la solución amortiguadora, consiste en una solución carbonato de sodio (100 mM) y dodecilsulfato sódico (SDS) al 10% (p/v) en solución con agua bidestilada con un pH en el rango entre 8 a 9].  a) precipitates using TC A / acetone [lg TCA / mL acetone, previously cooled to 4 ° C] and subsequently resuspended in a buffer solution [the buffer solution, consists of a sodium carbonate solution (100 mM) and sodium dodecyl sulfate (SDS ) at 10% (w / v) in solution with double-distilled water with a pH in the range between 8 to 9].
b) Ultrafiltradas para eliminar la solución no compatible y al mismo tiempo concentrar las proteínas y recuperarlas con la adición de solución amortiguadora que consiste en una solución de carbonato de sodio (100 mM) y dodecilsulfato sódico (SDS) al 10% (p/v) en solución con agua bidestilada, a un pH en el rango de 8 a 9.  b) Ultrafiltrates to eliminate the unsupported solution and at the same time concentrate the proteins and recover them with the addition of buffer solution consisting of a solution of sodium carbonate (100 mM) and 10% sodium dodecyl sulfate (SDS) ) in solution with double-distilled water, at a pH in the range of 8 to 9.
8. El método para modificación covalente de proteínas de conformidad con la reivindicación 1, caracterizado porque en la etapa a), en la solución colorante el grupo funcional para reaccionar en una adición nucleofilica es vinil. 8. The method for covalent modification of proteins according to claim 1, characterized in that in step a), in the dye solution the functional group for reacting in a nucleophilic addition is vinyl.
9. El método para modificación covalente de proteínas de conformidad con la reivindicación 1, caracterizado porque en la etapa a), en la solución colorante el grupo funcional ionizable para dar mayor solubilidad a la tinción en agua, posteriormente dar mayor solubilidad a los péptidos e influye su ionización por espectrometría de masas a los péptidos y solubilidad a la tinción en agua, preferentemente es sulfato. 9. The method for covalent modification of proteins according to claim 1, characterized in that in step a), in the dye solution the ionizable functional group to give greater solubility to water staining, then give greater solubility to the peptides and their ionization influences by mass spectrometry to the peptides and solubility to staining in water, preferably it is sulfate.
10. El método para modificación covalente de proteínas de conformidad con la reivindicación 1, caracterizado porque en la etapa g) la reacción de hidrólisis por tripsina cerca de residuos modificados con Uniblue A es inhibida, y debido a esto la reacción de hidrólisis se lleva a cabo en mayor parte en los residuos de arginina obteniendo fragmentos proteolíticos más grandes, logrando un mejoramiento en la cobertura de la secuenciación de péptidos. 10. The method for covalent modification of proteins according to claim 1, characterized in that in step g) the trypsin hydrolysis reaction near residues modified with Uniblue A is inhibited, and because of this the hydrolysis reaction is carried out carried out mostly in arginine residues obtaining larger proteolytic fragments, achieving an improvement in the coverage of peptide sequencing.
11. El método para modificación covalente de proteínas de conformidad con la reivindicación 1, caracterizado porque en la etapa j), los iones del grupo N-terminal precedidos por una lisina tienen una masa adicional correspondiente al Uniblue A (484.0 Da), lo que facilita su detección mediante espectrometría de masas. 11. The method for covalent modification of proteins according to claim 1, characterized in that in step j), the ions of the N-terminal group preceded by a lysine have an additional mass corresponding to Uniblue A (484.0 Da), which facilitates its detection by mass spectrometry.
12. El método para modificación covalente de proteínas de conformidad con la reivindicación 1, caracterizado porque en la etapa j), cada modificación de lisina afecta la masa de los fragmentos de aminoácidos en dirección N- o C- terminal. 12. The method for covalent modification of proteins according to claim 1, characterized in that in step j), each lysine modification affects the mass of the amino acid fragments in the N- or C-terminal direction.
13. El método para modificación covalente de proteínas de conformidad con la reivindicación 1, caracterizado porque en la etapa j), los iones del grupo N-terminal precedidos por al menos una lisina tienen una masa adicional acumulable correspondiente al Uniblue A (484.0 Da), respecto a la lisina. 13. The method for covalent modification of proteins according to claim 1, characterized in that in step j), the ions of the N-terminal group preceded by at least one lysine have an additional cumulative mass corresponding to Uniblue A (484.0 Da) , regarding lysine.
14. El método para modificación covalente de proteínas de conformidad con la reivindicación 1, caracterizado porque en la etapa j), los iones del grupo C-terminal precedidos por al menos una lisina tienen una masa adicional acumulable correspondiente al Uniblue A (484.0 Da), respecto a la lisina. 14. The method for covalent modification of proteins according to claim 1, characterized in that in step j), the ions of the C-terminal group preceded by at least one lysine have an additional cumulative mass corresponding to Uniblue A (484.0 Da) , regarding lysine.
15. Un kit para visualizar instantáneamente proteínas para su posterior caracterización usando espectrometría de masas, caracterizado porque comprende los siguientes reactivos almacenados de manera independiente: 15. A kit to instantly visualize proteins for subsequent characterization using mass spectrometry, characterized in that it comprises the following independently stored reagents:
a) Una solución amortiguadora A, que consiste en una solución de carbonato de sodio a una concentración 100 mM y una solución de dodecilsulfato sódico (SDS) a una concentración de 10% (p/v), con un rango de pH de 8 a 9; b) Solución de Colorante Uniblue A, en donde el Uniblue A es disuelto en la solución amortiguadora A, a una concentración de 60 mM; a) A buffer solution A, consisting of a solution of sodium carbonate at a concentration of 100 mM and a solution of sodium dodecyl sulfate (SDS) at a concentration of 10% (w / v), with a pH range of 8 to 9; b) Uniblue A Dye Solution, where Uniblue A is dissolved in buffer solution A, at a concentration of 60 mM;
c) Solución reductora, consiste en una solución al 20% (v/v) de glicerol, Tris HCl 200 mM y 20 mM ditiotreitol (DTT), con un pH de 6.8;  c) Reducing solution, consists of a 20% (v / v) solution of glycerol, 200 mM Tris HCl and 20 mM dithiothreitol (DTT), with a pH of 6.8;
d) Solución de alquilación, consiste en una solución de iodoacetamida (IAA) a una concentración de 550 mM en solución amortiguadora A;  d) Alkylation solution, consists of a solution of iodoacetamide (IAA) at a concentration of 550 mM in buffer solution A;
e) Control positivo: consiste en 3 mg de albúmina de suero bovino (ASB) liofilizada, con la instrucción para reconstituir en 1 mL de solución amortiguadora A.  e) Positive control: consists of 3 mg of lyophilized bovine serum albumin (ASB), with the instruction to reconstitute in 1 mL of buffer solution A.
16. Un kit para visualizar instantáneamente proteínas para su posterior caracterización usando espectrometría de masas, de conformidad con la reivindicación 15, caracterizado porque las instrucciones son: 16. A kit for instantly visualizing proteins for subsequent characterization using mass spectrometry, according to claim 15, characterized in that the instructions are:
a) Adicionar 10 μL· de solución colorante a 90 μL· de solución muestra que contiene la proteína de interés, para obtener una solución muestra coloreada, b) Calentar la solución muestra coloreada a 100 °C por 1 minuto,  a) Add 10 μL · of dye solution to 90 μL · of sample solution containing the protein of interest, to obtain a colored sample solution, b) Heat the colored sample solution at 100 ° C for 1 minute,
c) Agregar 100 μΐ.. de la solución reductora, a la solución muestra coloreada caliente, manteniendo el calentamiento a 100 °C por 1 minuto,  c) Add 100 μΐ .. of the reducing solution to the hot colored sample solution, keeping the heating at 100 ° C for 1 minute,
d) Agregar 20 \iL de la solución de alquilación a cada solución muestra coloreada caliente de c),  d) Add 20 µL of the alkylation solution to each hot colored sample solution of c),
e) Incubar la solución muestra coloreada de d) a temperatura ambiente por 5 minutos,  e) Incubate the colored sample solution of d) at room temperature for 5 minutes,
f) Cargar la solución muestras coloreada en el gel de SDS-PAGE,  f) Load the colored sample solution into the SDS-PAGE gel,
g) Separar las proteínas de la muestra coloreada de f) mediante electroforesis, opcionalmente a 150 V por 45 minutos, Bio-Rad Protean3 Minigel 10% Acrilamida.  g) Separate the proteins from the colored sample of f) by electrophoresis, optionally at 150 V for 45 minutes, Bio-Rad Protean3 Minigel 10% Acrylamide.
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