Automated Analysis of Hemoglobin Variants Using NanoLC–MS and

May 21, 2013 - In addition, unavailability of a variant database compatible with proteomics data analysis software makes mass spectrometry based varia...
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Automated Analysis of Hemoglobin Variants Using NanoLC−MS and Customized Databases Rajdeep Das,† Gopa Mitra,† Boby Mathew,† Cecil Ross,‡ Vijay Bhat,§ and Amit Kumar Mandal*,† †

Division of Molecular Medicine, Clinical Proteomics Unit, St. John’s Research Institute, St. John’s National Academy of Health Sciences, Bangalore 560034, India ‡ Department of Medicine, St. John’s Medical College and Hospital, St. John’s National Academy of Health Sciences, Bangalore 560034, India § Manipal Hospital, Old Airport Road, Bangalore, India S Supporting Information *

ABSTRACT: Unambiguous analysis of hemoglobin variants is critical in the diagnosis of hemoglobinopathies. In diagnostic laboratories, alkaline gel electrophoresis and automated HPLC are used in identifying variants. In specific instances, comigration of hemoglobin variant bands in gel and coelution of different variants or elution of variants with unmatched library information in HPLC can result in ambiguities in interpretation. Hemoglobin variants mostly arise from point mutations leading to very high sequence homology between normal and variant hemoglobin. In addition, unavailability of a variant database compatible with proteomics data analysis software makes mass spectrometry based variant analysis very challenging. In the present study, we standardized a nanoLC−MS based method for variant analysis to achieve substantially high sequence coverage. We developed three hemoglobin variant databases, specific to three different proteolytic enzymes, compatible with proteomics search engine software. The above nanoLC−MS method and the compatibility of the customized databases were validated by analysis of a sickle hemoglobin variant. Six other hemoglobin variants were characterized wherein diagnosis reports based on conventional tools were ambiguous. The novelty of our method lies in its simplicity and accuracy of the analysis with minimal manual intervention. The presently described method may be used in the future for the routine hemoglobin variant diagnosis. KEYWORDS: hemoglobin variant, mass spectrometry, database, nanoLC, D-10 HPLC

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cyanosis.10,11 There are many clinically silent hemoglobin variants as well, e.g., Hb Le Lamentin, Hb City of Hope, etc.12,13 More than a thousand hemoglobinopathies has been discovered to date.14 In a clinical diagnostic laboratory, gel electrophoresis and cation exchange chromatography (Bio-Rad HPLC) are used to identify hemoglobin variants. In both methods, tetrameric hemoglobin molecules are separated on the basis of their surface charges. In alkaline gel electrophoresis, hemoglobin variants are characterized according to their differential mobility in the gel. Comigration and/or unusual movement of hemoglobin bands in the gel results in ambiguity in the analysis.15 In cation exchange chromatography, variants are characterized by comparing their elution profile with the library-matched retention time for the variants.16 Coelution of different hemoglobin variants and elution with unmatched library retention times causes difficulty in the analysis.16 The

INTRODUCTION Hemoglobinopathies are genetic disorders caused by mutations in the globin gene.1 The mutants are termed as hemoglobin variants and characterized by the substitution/deletion/ insertion/fusion of amino acids in the globin polypeptide chains, exemplified by HbS, Hb Lincoln Park, Hb Catonsville, and Hb Lepore Boston Washington, respectively.2−5 Amino acid substitution in the hemoglobin may lead to structural changes in the molecule which in turn may affect its function. The associated functional abnormality may be displayed with a specific clinical phenotype. For example, in sickle hemoglobin HbS, substitution of glutamic acid with valine at the sixth position of the β globin chain (βE6V) causes hemoglobin polymerization that subsequently results in sickle cell anemia.2 In Hb Köln, βV98M mutation causes anemia due to formation of unstable hemoglobin.6 The increased oxygen affinity in Hb Betheseda results in erythrocytosis,7 HbC and Hb Raleigh have been reported to cause hemolytic anemia and decreased oxygen affinity, respectively. 8,9 Homozygous HbE shows mild hemolytic anemia and microcytosis, while HbM causes © XXXX American Chemical Society

Received: January 21, 2013

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where the collision energy was linearly ramped from 15 to 40 eV to fragment precursor ions. The instrument was operated in positive ion ‘V’ mode with a source temperature of 70 °C, capillary voltage of 2.8 kV, cone voltage of 32 V, and extraction cone voltage of 4 V. Reference compound Glu-fibrinopeptide B (GFP) was continuously infused through lockspray and scanned intermittently every 45 s. All samples were run in duplicate, and reproducible results were obtained with >95% confidence in PLGS search. MS calibration was done with GFP.

mass spectrometric characterization of hemoglobin variant is based on tandem MS analysis of proteolytic peptides generated by the digestion of globin chains. Due to very high sequence homology between normal and variant hemoglobin, the LC− MS based characterization of variants requires high sequence coverage. In addition, a database of hemoglobin variants compatible to proteomics search engine software is essential to process mass spectrometric data in an automated manner. In the present study, we developed a nanoLC−MS method and customized databases consisting of signature peptides for identification and characterization of hemoglobin variants with a high degree of precision. We validated our method with sickle hemoglobin. Six other hemoglobin variants were characterized where the results based on conventional methods were ambiguous.

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LC−ESI−MS Experiment

For intact globin chain mass analysis, 5 μg of hemolysate from variant sample was injected into a C18 RP column (Eclipse, 4.6 mm × 150 mm, 5 μm) in a Shimadzu UFLC coupled to Synapt HDMS. The data were acquired in positive ion “V” mode over mass range 650−1500 m/z, with capillary voltage of 3 kV using a source temperature of 120 °C and desolvation gas temperature of 350 °C. The different globin chains were separated using a linear gradient of 2% increase in acetonitrile per minute containing 0.1% acetic acid with a flow rate of 0.2 mL/min. The mass calibration was done using NaI. The mass spectrum was smoothed, baseline subtracted, and subsequently deconvoluted using MaxEnt1 software.

MATERIALS AND METHODS

Materials

LC−MS grade acetonitrile, water, and formic acid (FA) were purchased from Fluka (St. Louis, MO, USA). RapiGest, Sodium Iodide (NaI) and GFP were obtained from Waters (Milford, MA, USA). Proteolytic enzymes trypsin, chymotrypsin, and GluC were purchased from Sigma Aldrich (St. Louis, MO, USA). All other reagents used were analytical grade.

Database Design

The existing human hemoglobin variant database has 1153 entries to date.14 It provides information about the genetic mutations, changes in the amino acid, and clinical phenotype associated with the variants. However, this database is incompatible to proteomics search engine software for mass spectrometric data analysis. In this study, we developed three customized hemoglobin variant databases, HbVD Tryp , HbVDChymotryp, and HbVDGluC where each of the databases is specific to a particular proteolytic enzyme used in the digestion of globin chains. Each variant entry annotation was done following the conventional UniProtKB/Swiss-Prot database for human proteome. Instead of the entire globin chain sequence corresponding to a particular variant, the single proteolytic fragment characteristic to every individual variant was incorporated into the customized database. The signature peptide of a variant contains information about substitution/ deletion/insertion/fusion of amino acids in the particular globin subunit. The signature peptide was generated by theoretical digestion of the respective globin chain amino acid sequence with a specific proteolytic enzyme using Protein Prospector Software.17 In the database, each variant starts with an accession number, mnemonic organism identification code followed by hemoglobin subunit, variant name, nature and site of mutation, origin of species, gene name, protein existence level, and the signature peptide characteristic of the hemoglobin variant. The database was constructed with a unique identification code for each variant. For example, in the case of HbS, the substitution of E to V at the sixth position of the β globin chain was introduced as ‘E6V’. For Hb Lincoln Park, the deletion of valine at the 137th position of the δ globin chain was assigned as ‘V137’. Hb Catonsville, where valine was inserted between codons 37 and 38 in the α chain, was assigned as ‘37V38’ in the database. In Hb Lepore Boston Washington, δβ fusion between the 87th residue of the δ chain and 116th residue of the β chain was represented as ‘87delta116beta’. The database HbVDTryp was generated by incorporating signature peptides of variants generated using trypsin as the proteolytic enzyme with zero missed cleavage. Similarly, HbVDChymotryp and HbVDGluC were obtained using chymotrypsin and GluC

Sample Preparation

Venus blood, anticoagulated with EDTA, was collected from patients with hemoglobinopathies with their prior written consent from St. John’s Medical College Hospital and Manipal Hospital, Bangalore. After centrifugation at 3000 rpm for 10 min at 25 °C, the obtained packed cells were washed with 0.9% NaCl (aqueous) thrice before its lysis with eight volumes of icecold distilled water. The hemolysate was centrifuged at 12880g for 10 min at 4 °C to remove the erythrocyte membranes. Proteolytic Digestion

The isolated hemoglobin was digested with proteolytic enzymes trypsin, chymotrypsin, and GluC in three different sets following the procedure mentioned below. Hemoglobin was denatured by incubating 50 μg of protein with 8 μL of 0.2% RapiGest in 50 mM NH4HCO3, pH 7.8 at 80 °C for 15 min. Following denaturation, proteolytic digestion was performed at 37 °C for overnight using an enzyme to hemoglobin ratio of 1:10 (w/w). To breakdown Rapigest, the digested sample was acidified with 1 μL of FA and incubated at 37 °C for 90 min. It was then centrifuged at 6000 rpm for 20 min, and supernatant was taken for mass spectrometric analysis. NanoLC−MS Experiment

The proteolytic peptides were fractionated in NanoLC (NanoAcquity UPLC, Waters). 400 fmol of digested protein was loaded onto a NanoAcquityTP UPLC BEH C18 column (250 mm × 75 μm, 1.7 μm) at 35 °C. Peptides were eluted with a gradient of 0.5−40% of solvent B over 60 min using a 300 nL/min flow rate, where solvent A and B consisted of water and acetonitrile with 0.1% FA respectively. A trapping step of 1 min was used for desalting of sample in SymmetryR C18 column (180 μm × 20 mm, 5 μm). The eluted peptides were analyzed in Synapt HDMS (Waters, UK) coupled to NanoLC. The data were acquired using MSE mode of acquisition (mass range 50 −1600 m/z) that comprised of two mass scanning steps. In the first step, MS data were acquired at low collision energy (4 eV) to analyze peptide precursor ions. The second step was executed in MS mode B

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Figure 1. Mass spectrometric data analysis of tryptic digest of HbS sample. (A) The representative base peak chromatogram of tryptic peptides of HbS sample. The signature peptide eluted at 28.04 min (encircled peak). (B) Screenshot of PLGS analysis of HbS sample using HbVDTryp database. (C) Tandem mass spectrum of signature peptide with m/z 922.5, obtained from PLGS analysis. Series of “b” ions (blue), “y” ions (red), and ions obtained by the neutral losses (green) are shown.

nopathies is to screen population for hemoglobin variants. Routinely used methods of variants analysis such as gel electrophoresis and/or automated HPLC often provide ambiguous results due to comigration and/or coelution of the variants. Hence, it is important to establish a method that could identify and characterize all hemoglobin variants unambiguously. In recent years, mass spectrometry has been used in the identification and characterization of hemoglobin variants.20 Different intact globin polypeptide chains are spread across the mass range between 15100 Da to 16100 Da. Under electrospray ionization conditions, all globin molecular ions are distributed within the charge states +12 to +20.20 Thus, simultaneous detection of two globin chains with a unit mass difference requires a mass analyzer with a very high mass resolution. Excluding isobaric amino acid substitution, intact globin chain mass measurement provides information about the nature of the mutation present, though site-specific characterization is beyond the scope of this method. Mass spectrometry based proteomics has two strategic approaches, top-down and bottom-up. In the top-down approach, it is difficult to obtain the sequence of the entire polypeptide chain21,22 and thus is challenging to locate the point mutation. In the bottom-up mode, proteins are subjected to proteolytic digestion, and the corresponding peptides are analyzed by MS and tandem MS.23 Both MS and MS/MS data are analyzed using proteomics search engine software to identify protein through a database search. Hemoglobin variants mostly arise from the point mutation, leading to very high sequence homology between normal and variant globin chains. Hence, mass spectrometric variant analysis requires ∼100% sequence coverage. To achieve

enzymes respectively. All three customized databases (provided as supplement) were used in mass spectrometric data analysis and were found to be compatible to search engine software Protein Lynx Global Server (PLGS 2.5). Mass Spectrometric Data Analysis

Using an electrospray-MSE template, PLGS identifies a protein from the list of precursor ion and its corresponding fragment ions generated by an algorithm Apex-3D. In our analysis, a minimum of three fragment ions were selected to identify a peptide. Mass tolerances for precursor and product ions were set to automatic mode. During PLGS analysis, oxidation of methionine was allowed as a variable modification and missed cleavage was set to zero.

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RESULTS AND DISCUSSION Hemoglobinopathies comprise hemoglobin variants and thalassemia that affect the synthesis and the function of hemoglobin molecules in red blood cells (RBCs). Around 7% of the world populations are carriers of globin gene mutations.18 Though most of the hemoglobin variants are clinically silent, some of them are associated with clinical manifestations as mentioned previously. Thalassemia is characterized by reduced synthesis or complete absence of either the α or β globin chain. Coexistence of hemoglobin variants with thalassemia results in a severe condition; e.g, HbS with beta-thalassemia (HbβS/β0thal) and HbS with Lepore (HbβS/βLepore) lead to more intense sickling of erythrocytes.19 Again, co-occurrence of HbE with β thalassemia (HbβE/β0thal) is associated with an array of clinical complications.10 One viable option to prevent/control thalassemia and hemoglobiC

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Table 1. Bio-Rad D-10 and Mass Spectrometric Data of Hemoglobin Variants Bio-Rad D-10 sample Hb S 1 2 3 4 5 6

retention time (min)

% of variant

4.20 3.27 2.91 3.97 3.98 4.20 1.20 5.03

56.9 99.9 21.3 70.9 44.0 34.4 22.0 3.01

mass spectrometric results description

precursor ion MH+

tryptic signature peptide

description and interpretation

HbS HbA2 HbA2 unknown unknown HbS unknown unknown

922.5 916.4 1313.6 1377.6 1377.6 922.5 3238.6 2987.5

VHLTPVEK VNVDEVGGK VNVDQVGGEALGR QFTPPVQAAYQK QFTPPVQAAYQK VHLTPVEK VADALTNAVAHVDDMPNALSALSDLHAHTLR VADALTNAVAHVDDMPNALSALSDLQAHK

HbS β6E6V HbE βE26K HbD Iran βE22Q HbD Punjab βE121Q HbD Punjab βE121Q HbS βE6V HbJ Rajappen αK90T Hb Lansing αH87Q

In this study, we demonstrated our approach through the analysis of seven hemoglobin variant samples. We validated our method with sickle hemoglobin variant which had been unambiguously characterized by D-10 HPLC. The tryptic peptides of HbS were analyzed in the nanoLC−MS system using MSE mode of acquisition. Figure 1A depicts the representative base peak chromatogram of the sickle sample. The signature peptide (922.5 m/z) of HbS variant, VHLTPVEK (β1−8) (Charge 2+, precursor rms mass error 6.5 ppm, PLGS score 32772.3), eluted at 28.04 min in the nanoLC profile. The obtained mass spectrometric data were analyzed in PLGS 2.5 software using HbVDTryp database. Figure 1B shows PLGS output where the sample was assigned as HbS, on the basis of the signature peptide identified at 922.5 m/z. Figure 1C shows MS/MS spectrum of the signature peptide, obtained as PLGS output. PLGS result of HbS sample is summarized in Table 1. Using the nanoLC−MSE method, we analyzed six experimental hemoglobin variant samples which appeared with either abnormally elevated level of HbA2 or at least one unassigned peak in D-10 HPLC. In D-10 HPLC, sample-1 was assigned with 99.9% abundance of HbA2. In general, the HbA2 level in β-thalassemia is less than 7%.28 In D-10 HPLC, a few variants such as HbE, HbLepore, polyadenylation coelute with HbA2 and provide a falsely high percentage of HbA2. HbE heterozygotes and homozygotes were reported to have an average elevated HbE of 30% and 84% respectively.29 Under such circumstances, the variant identification by D-10 HPLC is only presumptive, and it needs to be cross checked by other methods. In addition, due to comigration, alkaline gel electrophoresis is unable to distinguish between HbA2 and HbE.30 NanoLC−MSE analysis of tryptic peptides of sample-1 followed by PLGS analysis using the HbVDtryp database identified the sample as HbE explicitly with the signature peptide VNVDEVGGK (β18−26) at 916.4 m/ z (Charge 2+, precursor rms mass error 4.1 ppm, PLGS score 20691.5) and mutation βE26K (Table 1). D-10 HPLC analysis showed that sample-2 had 21.3% HbA2. Trypsinized sample-2 was subjected to nanoLC−MSE analysis, and the obtained data were processed in an identical manner as followed for sample-1. The sample was identified as HbD Iran with signature peptide at 1313.6 m/z corroborating the sequence VNVDQVGGEALGR (β18−30) (Charge 2+, precursor rms mass error 1.1 ppm, PLGS score 75953.7) and mutation βE22Q (Table 1). Previously, we reported that due to coelution, HbD Iran and HbA2 variants cannot be distinguished in D-10 HPLC.30 Thus, the mass spectrometry based method seems to be a better choice to differentiate the two aforementioned variants.

substantially high sequence coverage, we standardized a nanoLC−MS method where proteolytic peptides were fractionated extensively. In the second step, both MS and MS/MS information of above proteolytic peptides were obtained through MSE mode of acquisition. Finally, the obtained MSE informations were analyzed using a customized database consisting of signature peptides of the hemoglobin variants. Previously, Basilico et al. reported hemoglobin variant characterization using a data-dependent (DDA) MS scan where the three most abundant peptides per MS scan were fragmented.24 The DDA method has limitation over selecting the number of peptides in the MS/MS scan.25,26 Because of the competition in the ionization probability of peptides and limitation in the fractionation efficiency of LC, it is uncertain that the signature peptide would always be present within the three most abundant peptides in the LC−MS analysis of the proteolytic digest of the hemoglobin variant. Thus, there are chances of missing the signature peptide in the method, which can select only a limited number of peptides for MS/MS scan.25,26 In the presently described data independent (MSE) method, every peptide ion coming across the MS scan was subjected to fragmentation in the subsequent high energy MS scan, thus minimizing the possibility of missing the signature peptide in the MS/MS scan.25 Additionally, the DDA mode can be executed including the signature peptide mass in the MS/ MS scan list.27 This strategy is feasible for samples where the nature and site of mutation is well-known. Therefore, compared to DDA, the data independent MSE scan might be a better method to characterize hemoglobin variants. The previous study was based on trypsin digestion of the variant sample. A closer look at the database revealed that due to specificity of the cleavage site at the C-terminus of lysine and arginine, trypsin may not generate signature peptide for some of the hemoglobin variants, e.g., Hb Clinic, which is characterized by the deletion of αK61 residue out of two consecutive lysine residues, αK60K61, in the α globin chain. GluC might be a more suitable proteolytic enzyme for Hb Clinic analysis, as the chymotryptic signature peptide (5338.7 Da) is too large to sequence. Thus, occurrence of very few and/or too many amino acids in the signature peptide might cause difficulty in variant analysis. These issues might be overcome by using multiple proteolytic enzymes. In addition, the database used in the earlier study consisted of tryptic peptides of the hemoglobin variants originated from α and β globin chains,24 thus ignoring the possibilities of variants originating from δ and γ globin chains. Our customized databases include hemoglobin variants from all globin chains. D

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Figure 2. Mass spectrometric data analysis of tryptic digest of sample-5. (A) BioRAD D-10 HPLC profile of sample-5 where the peak eluted at 1.20 min was assigned as unknown. (B) Screenshot of PLGS analysis of sample-5 using the HbVDTryp database where the variant was identified as HbJ Rajappen. (C) Tandem mass spectrum of the signature peptide with m/z 3238.6, obtained from PLGS analysis. Series of “b” ions (blue) and “y” ions (red) are shown.

PLGS score 1845.7). The mass spectrometric analysis report is summarized in Table 1. Sample-6 was assigned with an unknown peak at 5.03 min in D-10 HPLC. Figure 3A shows the base peak chromatogram of trysinized sample-6 obtained in the nanoLC−MSE experiment. Subsequent data analysis identified the sample as Hb Lansing on the basis of signature peptide VADALTNAVAHVDDMPNALSALSDLQAHK (α62−90) at 2987.50 m/z (Charge 4+, precursor rms mass error 5.8 ppm, PLGS score 12390.8) and αH87Q mutation (Table 1). The PLGS output and the MS/ MS spectrum of signature peptide are shown in Figure 3B and 3C, respectively. Recently, Hb Lansing has been reported for the first time in Asia.32 To reconfirm the variant, sample-6 was subjected to intact globin chain mass analysis using LC−ESI− MS following the procedure described by Mandal et al.33 Figure 4A and 4B show the total ion chromatogram and mass spectrum of intact globin chains, respectively, obtained from sample-6. Figure 4C represents the deconvoluted mass spectrum. Both charge state distribution (Figure 4B) and deconvoluted mass spectrum indicated the presence of an α variant globin chain along with the normal α globin chain. The mass difference between variant and normal α chain was found to be 9 Da, which could be assigned to the mutation αH → Q, identifying the sample-6 as Hb Lansing. We validated the compatibility of HbVDchymotryp database and PLGS through the analysis of chymotryptic digest of sickle sample where HbS was identified with signature peptide 1650.9

D-10 HPLC profiles of sample-3 and sample-4 had been assigned with an unknown peak eluting at 3.97 min and 3.98 min, respectively. Additionally, sample-4 had the characteristic peak for HbS. PLGS analysis of nanoLC−MSE data for sample3 and sample-4 using the HbVDTryp database identified the presence of HbD Punjab variant in both the samples. The variant was assigned on the basis of the signature peptide QFTPPVQAAYQK (β121−144) at 1377.6 m/z (Charge 2+, precursor rms mass error 2.4 ppm, PLGS score 20482.3) with mutation βE121Q (Table 1). Because of the presence of signature peptide VHLTPVEK at 922.5 m/z (Charge 2+, precursor rms mass error 4.7 ppm, PLGS score 46125.7), sample-4 was also assigned as HbS (Table 1). Comigration of HbD Iran and HbD Punjab variants in alkaline gel makes it difficult to distinguish them in electrophoresis. Therefore, the presently described technique might offer a convenient screening tool for variants such as HbD Iran, HbD Punjab, and HbA2 where both D-10 HPLC and/or alkaline gel electrophoresis provide ambiguous results. For sample-5, the D-10 HPLC profile displayed an unknown peak at 1.20 min, as depicted in Figure 2A. The mass spectrometric analysis of tryptic peptides of sample-5 identified the variant as HbJ Rajappen. Figure 2B shows PLGS output of sample-5 as HbJ Rajappen with mutation αK90T.31 Figure 2C represents the MS/MS spectrum of the signature peptide VADALTNAVAHVDDMPNALSALSDLHAHTLR (α62−92) at 3238.7 m/z (Charge 5+, precursor rms mass error 7.2 ppm, E

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Figure 3. Mass spectrometric analysis of tryptic digest of sample-6. (A) The representative base peak chromatogram of tryptic peptides of sample-6. The signature peptide eluted at 56.03 min (encircled peak). (B) Screenshot of PLGS analysis of sample-6 using HbVDTryp database where the variant was identified as Hb Lansing. (C) Tandem mass spectrum of the signature peptide with m/z 2987.5, obtained from PLGS analysis. Series of “b” ions (blue) and “y” ions (red) are shown.

chains. Two β variants, Hb Cambridge MA and Hb Mckees Rocks, and one α variant Hb Natal originated by the deletion of C-terminal residues KYH, YH and YR from respective globin chains. To remove artifacts in PLGS analysis report, three above variants were removed from the customized databases. The variant HbP Nilotic originated by the fusion of the 30th residue of β globin and the 31st residue of δ globin chains. As the 30th residue of β globin chain is lysine, the tryptic signature peptide cannot be generated for HbP Nilotic. We have already mentioned that for Hb Clinic tryptic signature peptide cannot be generated. Hence, the above two variants were removed from the HbVDTryp database, though those two variants can be analyzed unambiguously using HbVDchymotryp, HbVDGluCdatabases. Intact globin chain mass measurements might provide an indication of the presence of above deleted variants. There are 36 hemoglobin variants discovered to date carrying two mutations in the same globin chain. Nine of them have tryptic signature peptides consisting of both the mutations and have been incorporated in HbVDTryp with their signature sequences. Four of these double mutants have at least one unique tryptic signature peptide and are included in the database accordingly. Rest of the 23 variants have both the signature peptides identical to other hemoglobin variants, and the present method of analysis might provide ambiguous results for these variants. Thus, those variants were removed from the HbVDTryp database. Similar strategies have been followed while constructing HbVDChymotryp and HbVDGluC databases for double mutants.

m/z (β1−15). Similarly, analysis of GluC digest of HbS and HbE variants using the HbVDGluC database identified it successfully with signature peptides 794.4 m/z (β1−7) and 2436.3 m/z (β23−43) for HbS and HbE respectively (data not shown). Initially, we observed that irrespective of the type of experimental sample, the PLGS analysis report always consisted of a few variants along with the variant of interest. A close review of the result revealed that the signature peptides of those variants had matched the proteolytic fragment originating from other normal globin chains. For example, in the case of Hb Flatbush, a δ variant with the mutation δA22E, the signature tryptic peptide “VNVDEVGGEALGR” was identical with the tryptic peptide β18−30 of the normal β globin chain. For Hb Charlotte, a γA variant with mutation γAA136G, the signature tryptic peptide was identical in its sequence with the tryptic peptide γ133−144 of the normal γG globin chain. A similar situation occurred in case of Hb Coushatta (βE22A) and Hb Zurich langestaresse (βT50S), where signature peptides were identical to the normal δ globin tryptic peptide fragments δ18−40 and δ41−59 respectively. However, the choice of different proteolytic enzymes did not improve the situation. Therefore, to remove the artificial ambiguity in PLGS analysis, the above four variants were removed from three customized databases. Again, in addition to the signature peptide, PLGS analysis report of any hemoglobin variant was found to consist of fragments of signature peptide generated at the MS source, known as in-source fragments. Hence, the variants originated by the deletion of amino acid residues from the terminus of globin chains were displayed in the PLGS report by default due to the occurrence of in-source fragmentation of normal globin F

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Figure 4. Intact globin chain mass analysis of sample-6. (A) Total ion chromatogram of sample-6 obtained in LC−ESI−MS experiment. (B) ESI mass spectrum of the sample where the charge states for different intact globin chains are labeled. (C) Deconvoluted mass spectra of normal Hb α chain (15126.2 Da), mutated Hb α chain (15117.6 Da), normal Hb β chain (15867.0 Da) are shown.

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To summarize, out of 1153 hemoglobin variants, the presently described method can be used to unambiguously characterize 1123 variants using HbVDTryp. Intact globin chain mass measurements may provide an indication for the presence of rest of the 30 variants. The proposed method takes a couple of hours for one variant analysis to complete. Being a mass spectrometry based proteomics analysis experiment, the described method is relatively expensive.

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ASSOCIATED CONTENT

S Supporting Information *

Details on HbVDTryp, HbVDChymotryp, and HbVDGluC. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Address: Clinical Proteomics Unit, Division of Molecular Medicine, St. John’s Research Institute, St. John’s National Academy of Health Sciences, 100 ft Road, Koramangala, Bangalore 560034, India. Phone: +91-80-25532037, Fax: +9180-25501088, E-mail: [email protected].

CONCLUSION

Analysis of hemoglobin variants using the nanoLC−MSE method and customized peptide database has been found to be rapid and robust with minimal manual intervention. Sometimes hemoglobin variant analysis using Bio-Rad D-10 and/or alkaline gel electrophoresis is found to be misleading. Presently, the described mass spectrometry based method provides unambiguous identification of hemoglobin variant with the nature and site of mutation in a single run. The novelty of the method lies in its simplicity, automation, and accuracy of the analysis. In the future, the described method may be used in the diagnosis of hemoglobinopathies.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge Department of Science & Technology (DST), Government of India, for funding the mass spectrometric facility at St. John’s Research Institute. We acknowledge patients who provided samples in the study. R.D. was supported by Senior Research Fellowship from Indian Council of Medical Research (ICMR), Government of India. The G

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Journal of Proteome Research

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authors thank Dr K Srinivasan, Dean, SJRI, for helpful discussions in manuscript preparation.

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ABBREVIATIONS USED GFP, Glu-fibrinopeptide B; RBC, red blood cell; PLGS, protein lynx global server; DDA, data-dependent analysis; nanoLC, nano liquid chromatography; MS, mass spectrometry; MSE, mass spectrometry in elevated energy

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