WO2011095981A1 - Procédé in vitro pour la caractérisation de variant d'hémoglobine - Google Patents

Procédé in vitro pour la caractérisation de variant d'hémoglobine Download PDF

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Publication number
WO2011095981A1
WO2011095981A1 PCT/IN2010/000217 IN2010000217W WO2011095981A1 WO 2011095981 A1 WO2011095981 A1 WO 2011095981A1 IN 2010000217 W IN2010000217 W IN 2010000217W WO 2011095981 A1 WO2011095981 A1 WO 2011095981A1
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Prior art keywords
hemoglobin
maldi
variant
gel
digest
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PCT/IN2010/000217
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English (en)
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Amit Kumar Mandal
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St. John's Research Institute
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
    • G01N27/4473—Arrangements for investigating the separated zones, e.g. localising zones by electric means
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
    • G01N33/721—Haemoglobin

Definitions

  • the present disclosure is related to a method of characterizing a sample. More essentially, the present disclosure provides an in-vitro method of characterizing hemoglobin variant using alkaline gel electrophoresis and MALDI-MS in tandem.
  • gel electrophoresis is the most commonly used method for hemoglobin variant analysis.
  • alkaline gel electrophoresis is a method followed for detecting common hemoglobin variants such as HbA, HbS, HbF, HbC.
  • HbS, HbD, HbG and HbLepore are difficult to resolve from each other on the basis of band position in gel. Similar difficulty exists in reference to resolution of HbC, HbA2, HbO- Arab and HbE.
  • HbS and HbC have similar electrophoretic mobility compared to few other variants which eventually results difficulty in characterization. In such a scenario, acid electrophoresis is a better choice.
  • HPLC high-performance liquid chromatography
  • cation exchange HPLC variants are separated on the basis of its affinity to polyaspartic acid coated stationary phase whereas in reverse phase HPLC (RP-HPLC) the separation is based on the hydrophobic ity of hemoglobin chains.
  • Automated HPLC system for Hb variant analysis has a library of retention times for different variants. However, appearance of a peak with unmatched library retention time creates difficulty in characterizing the variant. Also, co-elution of different variants also results in improper characterization, e.g., co- elution of HbE with HbA2 may result in elevated HbA2 values.
  • LC/ESI-MS has been used in hemoglobin variant analysis. These procedures need a highly efficient LC system like Nano-LC to fractionate proteolytic peptides extensively and expertise to execute the analysis successfully. Proteolytic peptides can also be fractionated in RP- HPLC and collected fractions can be analyzed individually using MALDI-MS and MALDI-MS-MS for subsequent sequencing of peptides. In above mentioned methods fractionation of sample is very much crucial. All the above proposed methods are expensive as well. Nonetheless, LC-ESI-MS method requires highly efficient chromatographic system like Nano-LC and skilled expertise is required to execute the analysis successfully. Again Nano-LC and Nano-LC-columns are very expensive. All these constitute error/ bias in the analysis.
  • hemoglobin variant identification is mostly PCR based method followed by confirmation of mutation through DNA sequencing. This method involves a large number of pre analytical steps to execute.
  • the present disclosure provides an in-vitro method for characterizing hemoglobin variant, comprising steps of: performing alkaline gel electrophoresis of sample to obtain hemoglobin bands; excising and digesting the hemoglobin gel bands to obtain a digest; and analyzing the digest by MALDI-MS followed by sequencing using MALDI-MS-MS to characterize the hemoglobin variant.
  • Panel A shows MALDI-MS spectra of tryptic peptides obtained from in-gel digestion of HbD band in alkaline gel electrophoresis.
  • Panel B shows MALDI-MS- MS spectra of the peptide ion with m/z 1377.7.
  • Panel A shows MALDI-MS spectra of chymotryptic peptides obtained from in- gel digestion of HbE band in alkaline gel electrophoresis.
  • Panel B shows MALDI- MS-MS spectra of the peptide ion with m/z 1285.7.
  • the present disclosure is in relation to an in-vitro method for characterizing hemoglobin variant, comprising steps of performing alkaline gel electrophoresis on sample to obtain hemoglobin bands; excising and proteolytically digesting the hemoglobin gel bands to obtain proteolytic peptides; and analyzing the peptides by MALDI-MS. Identifying the peptide characteristic to Hb variant followed by MALDI- MS-MS of the peptide for subsequent sequencing to characterize the hemoglobin variant.
  • the sample used for characterization of hemoglobin variants is a biological sample having concentration of hemoglobin at a concentration of about 3.0 gm/dL.
  • the alkaline agar gel bands are excised and are subjected to washing with wash solution comprising ammonium bicarbonate at a concentration ranging from about 40 mM to about 60 mM, preferably about 50mM and acetonitrile (ACN) at a concentration ranging from about 40 % v/v to about 60 % v/v, preferably about 50 % v/v.
  • wash solution comprising ammonium bicarbonate at a concentration ranging from about 40 mM to about 60 mM, preferably about 50mM and acetonitrile (ACN) at a concentration ranging from about 40 % v/v to about 60 % v/v, preferably about 50 % v/v.
  • ACN acetonitrile
  • the excised alkaline agar gel after washing is subjected to vigorous shaking for a time period ranging from about 1 minute to about 3 minutes, preferably for a time period of about 2 minutes.
  • digestion is performed using enzymes selected from a group comprising trypsin, chmotrypsin and endoproteinase Glu-C.
  • the protease enzyme to hemoglobin ratio is about 1 :50 w/w.
  • the digest is mixed with the matrix solution in a ratio of about 1 : 1 v/v followed by spotting on a MALDI plate.
  • the sequencing is performed by manual assignment of daughter ions.
  • identifying the peptide characteristic to Hb variant and is followed by MALDI-MS-MS for subsequent sequencing of the peptide fragment to characterize the site and nature of mutation in hemoglobin variant.
  • MALDI-MS matrix assisted laser desorption ionization mass spectrometry
  • Figl - Panel A shows MALDI-MS spectra of tryptic peptides obtained from in-gel digestion of HbD band obtained in alkaline gel electrophoresis.
  • Panel B shows MALDI-MS-MS spectra of the peptide ion with m/z 1377.7. Series of 'b', 'y' ions and neutral loses are labeled. The sequence obtained using de novo sequencing technique is shown as inset.
  • Fig 2 - Panel A shows MALDI-MS spectra of chymotryptic peptides obtained from in-gel digestion of HbE band obtained in alkaline gel electrophoresis.
  • Panel B shows MALDI-MS-MS spectra of the peptide ion with m/z 1285.7. Series of 'b', 'y' ions and neutral loses are labeled. The sequence obtained using de novo sequencing technique is shown as inset
  • Venus blood sample was collected in EDTA coated vaccutainer tube.
  • the vaccutainer tube was centrifuged at 3000 rpm for 5 minutes at 25°C.
  • the obtained packed cells were washed twice with 0.9% saline and were lysed with hemoglobin lysing reagent provided in electrophoresis kit (SAS-MX Alkaline Hb-10) supplied by Helena Bioscience.
  • the final concentration of hemoglobin in the lysate was 3.0 gm/dL.
  • the sample as obtained in example 1 is subjected to alkaline gel electrophoresis, which was performed in SAS-MX chamber electrophoresis system (Helena Bioscience, Europe) and alkaline electrophoresis kit (SAS- MX Alkaline Hb-10, Helena).
  • SAS-MX chamber electrophoresis system Helena Bioscience, Europe
  • SAS- MX Alkaline Hb-10, Helena alkaline electrophoresis kit
  • the excess moisture of the gel was gently blotted with a single sheet of gel blotter along the solid support.
  • a 10-slot sample application template was placed on the gel and aligned with the arrows at the edges of the gel. After loading 3 ⁇ 1 of hemolysate onto the corresponding template slots the diffusion was accomplished by keeping the gel for 5 minutes.
  • the gel was placed into the electrophoretic cell containing 40ml of Tris- Borate buffer, pH 8.5 (Helena) and electrophoresed at room temperature for 30 minutes at 150V.
  • the different bands obtained in the gel were fixed by immersing the gel in fixative solution (5:5:1 Methanol: Distilled water: Glacial acetic acid) for 5 minutes.
  • fixative solution 5:5:1 Methanol: Distilled water: Glacial acetic acid
  • the hemoglobin bands obtained as per example 2 were carefully excised from the alkaline agar gel and were minced into fine pieces in separate sets. Individual set was placed in a 0.5 ml centrifuge tube. The stain and salt were removed by washing with ⁇ of wash solution containing 50% ACN (v/v), 50mM NH 4 HCO 3 (aqueous) with vigorous shaking for 2 minutes followed by incubating at room temperature for 15 minutes. The above step was repeated until the gel was completely free from any traces of dye. Dehydration of the gel pieces was accomplished by adding 50 ⁇ , ⁇ of 100% ACN for 5 minutes.
  • In-gel digestion was performed using proteases like trypsin (TPCK treated, Sigma) or chymotrypsin (TLCK treated, Sigma) in 50mM NH 4 HCO 3 (aqueous) pH 8.0. The ratio of enzyme to hemoglobin was maintained at 1:50 (w/w) and incubated at 37°C for 12 hours.
  • Proteolytic peptides were eluted from gel pieces with ⁇ , elution buffer consists of 60% ACN (v/v), 1% TFA (aqueous) followed by sonication for 10 minutes. The above solution was centrifuged at 6000rpm for 1 min at 4°C and supernatant, consists of peptides, were collected. The above step was repeated once again to collect any residual peptides in gel.
  • the digest as obtained using example 3 is subjected to mass spectrometry.
  • the MALDI-MS for tryptic peptides is recorded using Synapt HDMS system (Waters, UK) in positive ion mode within a range of 900-3500m/z.
  • the Mass spectrometer was calibrated using an external calibrant, pegmix (Waters, UK) in above m/z range.
  • the mass spectra were acquired by integrating an average 40 shots/spot with a solid state LASER of wavelength 354 nm and analyzed using Masslynx V4.1 software.
  • the digested peptides were mixed with the matrix solution, a-cyano-4-hydroxy-cinnamic acid at 1:1 v/v ratio and spotted on MALDI plate. Tandem Mass spectra of the selected precursor ion was acquired with 15Da window using laser energy of 250Hz. De novo sequencing was done by manual assignment of daughter ions.
  • Example 5 Performing sample analysis using alkaline gel electrophoresis and MALDI-MS in tandem
  • the venous blood sample [healthy subjects, subjects with thalassemia or anemia] was collected in EDTA coated tubes and subjected to sequence of steps as recited under example 1.
  • alkaline gel electrophoresis was used where different variants appeared as distinct bands. It was performed using SAS-MX chamber electrophoresis system [Helena Bioscience, Europe] and alkaline electrophoresis kit (SAS-MX Alkaline Hb-10, Helena). Hemolysate obtained from different hemoglobin samples were loaded [3 ⁇ ] in corresponding wells and electrophoresed with Tris-Borate Buffer, pH 8.5 at 150V for 30 minutes, at room temperature. After fixing, the hemoglobin bands were stained using Acid blue stain (Helena) for 10 minutes followed by destaining for 25 minutes. More details are provided in example 2.
  • hemoglobin gel bands were excised.
  • the stain and salt were removed by washing with ⁇ , of wash solution containing 50% ACN (v/v), 50mM NH4HC03 (aqueous) with vigorous shaking for 2 minutes followed by incubating at room temperature for 15 minutes. Dehydration of the gel pieces was accomplished with 100 % ACN for 5 minutes.
  • In-gel digestion was performed using proteases like trypsin [TPCK treated, Sigma] in 50 mM NH4HC03, pH 8.0. The ratio of enzyme to hemoglobin was maintained at 1:50 (w/w) and incubated at 37°C for 12hrs.
  • Proteolytic peptides were eluted from gel pieces with elution buffer [60% ACN (v/v), 1% TFA (aqueous)].
  • the MALDI-MS spectra for tryptic peptides were recorded as explained in example 4.
  • the mass spectra were acquired by integrating an average 40 shots/spot with a solid state LASER of wavelength 354 nm and analyzed using Masslynx V4.1 software (Waters, UK). Tandem mass spectra of the selected precursor ion was acquired with 15Da window using laser energy of 250Hz. De novo sequencing was done by manual assignment of daughter ions.
  • the present disclosure provides a novel method of hemoglobin variant characterization and identification of mutation by coupling gel electrophoresis and MALDI-MS.
  • the highlights of the instant disclosure are provided as below:
  • the variant hemoglobin bands have been proteolytically digested and analyzed in MALDI-MS and MALDI-MS-MS.
  • HbA band digestion Analysis of tryptic peptides obtained from HbA band digestion showed sequence coverage of 75% and 70.5% for ⁇ and a globin chains of hemoglobin respectively. Similarly, HbF analysis exhibited 65% coverage for ⁇ chains. For a patient sample with elevated HbA 2 we got 68% coverage for the delta chain.
  • the peptide mass finger print for HbS band showed characteristic tryptic peptide with m/z 922.6 which is signature peptide to sickle cell mutation ⁇ E(6) to V.
  • the present disclosure provides analysis of two other hemoglobin variant samples that were characterized as HbD and HbE by BioRad-DIO machine.
  • HbE band MALDI-MS spectra of chymotryptic digested HbE band was compared with that obtained for HbA band digest for sample 2.
  • HbE band a monoisotopic peak appeared at 1285.7 m/z with simultaneous disappearance of a peak at 1286.7 m/z obtained in HbA digest (Fig 2, Panel A).
  • the tandem mass spectrum of precursor ion with m/z 1285.7 consists of series of 'b' and 'y' ions along with their neutral loses (Fig. 2 Panel B). Assignment of those daughter ions confirm that the peptide fragment corresponds to the residue 16 to 28 of ⁇ chain with the site of mutation E(26) to K (shown as insert in Panel B, Fig. 2).

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Abstract

La présente invention concerne un protocole simple et économique pour la caractérisation de variants d'hémoglobine. Il utilise une combinaison de techniques analytiques telles que l'électrophorèse sur gel en milieu alcalin et la spectrométrie de masse à désorption-ionisation laser assistée par matrice (MALDI-MS) et la spectrométrie de masse en tandem (MS-MS) en combinaison pour la caractérisation de variants d'hémoglobine. Le procédé selon la présente invention est rapide et efficace.
PCT/IN2010/000217 2010-02-03 2010-04-01 Procédé in vitro pour la caractérisation de variant d'hémoglobine WO2011095981A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109444279A (zh) * 2018-11-06 2019-03-08 北京蛋白世界生物科技有限公司 一种15n代谢标记蛋白质结合质谱平行反应监测定量的方法
CN111638261A (zh) * 2020-04-17 2020-09-08 融智生物科技（青岛）有限公司 一种计算设备、存储介质和地中海贫血筛查装置及系统

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WO2008098734A1 (fr) * 2007-02-12 2008-08-21 Stefan Hansson Diagnostic et traitement de la prééclampsie

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RAYMACKERS J ET AL.: "Identification of two-dimensionally separated human cerebrospinal fluid proteins by N-terminal sequencing, matrix-assisted laser desorption/ionization-mass spectrometry, nanoliquid chromatography-electrospray ionization-time of flight-mass spectrometry, and tandem mass spectrometry", ELECTROPHORESIS, vol. 21, 2000, pages 2266 - 2283, XP008001622, DOI: doi:10.1002/1522-2683(20000601)21:11<2266::AID-ELPS2266>3.0.CO;2-Z *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109444279A (zh) * 2018-11-06 2019-03-08 北京蛋白世界生物科技有限公司 一种15n代谢标记蛋白质结合质谱平行反应监测定量的方法
CN111638261A (zh) * 2020-04-17 2020-09-08 融智生物科技（青岛）有限公司 一种计算设备、存储介质和地中海贫血筛查装置及系统
CN111638261B (zh) * 2020-04-17 2023-04-07 融智生物科技（青岛）有限公司 一种计算设备、存储介质和地中海贫血筛查装置及系统

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