|Year : 2015 | Volume
| Issue : 1 | Page : 45-53
Identification of differentially expressed proteins in vitamin B 12
Swati Varshney1, Nitin Bhardwaj2, Trayambak Basak1, Shantanu Sengupta1
1 Department of Genomics and Molecular Medicine Unit, CSIR-Institute of Genomics and Integrative Biology, Sukhdev Vihar, New Delhi; Academy of Scientific and Innovative Research, CSIR-IGIB Campus, New Delhi, India
2 Department of Genomics and Molecular Medicine Unit, CSIR-Institute of Genomics and Integrative Biology, Sukhdev Vihar, New Delhi, India
|Date of Web Publication||22-May-2015|
Dr. Shantanu Sengupta
CSIR-Institute of Genomics and Integrative Biology, Mathura Road, New Delhi - 110 020
Source of Support: None, Conflict of Interest: None
Background: Vitamin B 12 (cobalamin) is a water-soluble vitamin generally synthesized by microorganisms. Mammals cannot synthesize this vitamin but have evolved processes for absorption, transport and cellular uptake of this vitamin. Only about 30% of vitamin B 12 , which is bound to the protein transcobalamin (TC) (Holo-TC [HoloTC]) enters into the cell and hence is referred to as the biologically active form of vitamin B 12 . Vitamin B 12 deficiency leads to several complex disorders, including neurological disorders and anemia. We had earlier shown that vitamin B 12 deficiency is associated with coronary artery disease (CAD) in Indian population. In the current study, using a proteomics approach we identified proteins that are differentially expressed in the plasma of individuals with low HoloTC levels. Materials and Methods: We used isobaric-tagging method of relative and absolute quantitation to identify proteins that are differently expressed in individuals with low HoloTC levels when compared to those with normal HoloTC level. Results: In two replicate isobaric tags for relative and absolute quantitation experiments several proteins involved in lipid metabolism, blood coagulation, cholesterol metabolic process, and lipoprotein metabolic process were found to be altered in individuals having low HoloTC levels. Conclusions: Our study indicates that low HoloTc levels could be a risk factor in the development of CAD.
Keywords: Holo-transcobalamin, isobaric tags for relative and absolute quantitation, plasma, proteomics, vitamin B 12
|How to cite this article:|
Varshney S, Bhardwaj N, Basak T, Sengupta S. Identification of differentially expressed proteins in vitamin B 12. J Pract Cardiovasc Sci 2015;1:45-53
|How to cite this URL:|
Varshney S, Bhardwaj N, Basak T, Sengupta S. Identification of differentially expressed proteins in vitamin B 12. J Pract Cardiovasc Sci [serial online] 2015 [cited 2018 Nov 18];1:45-53. Available from: http://www.j-pcs.org/text.asp?2015/1/1/45/157568
| Introduction|| |
Vitamin B 12 plays an essential role in the normal growth and development. Any defect in the genes involved in its absorption and transport can alter the vitamin B 12 levels leaving a profound effect on the physiology. Thus, it is important to study the basic perturbations occurring at the proteome level of healthy individuals with low and high HoloTC levels, which will help in understanding and delineating the importance of vitamin B 12 in complex disorders.
Proteins are the structural and functional unit of the cell. These are large biological macromolecules consisting of amino acid residues. Proteins perform a vast variety of functions ranging from supporting cell's cytoskeleton such as muscle proteins myosin and actins, to very sensitive roles of driving biochemical reactions like enzymes. , Classically studies have focused on single proteins but with the advent of mass spectrometry (MS) based techniques it is now possible to study large number of proteins simultaneously using proteomics approaches. , Proteomics can be defined as the global study of proteins, particularly their structure and function in a spatiotemporal context.  The term proteomics was coined by Mark Wilkins in 1990 when two terms proteins and genomics were taken together. , Proteomics is considered as an important tool of the postgenomics era. It has the ability to qualitatively and quantitatively identify proteins and scan the whole proteome, including protein expression, their cellular localization, protein-protein interactions, posttranslational modifications in space, time and cell type-dependent manner.  Proteomics is now playing a significant role in biomarker and drug target discovery for several diseases. 
The first protein studies that can be termed "proteomics" began with the introduction of two-dimensional (2D) gel electrophoresis where proteins could be separated and visualized, but could not be identified.  However, with the advancement of MS-based methods proteins can now be identified and quantified globally in-conjunction with several chromatography based methods.  In contrast to the genome, which is relatively static and essentially identical in every cell of an organism, protein expression is in a state of dynamic flux, constantly changing to external and internal stimuli. Further challenges are posed by obtaining sufficient quantities of a target protein from its biological source as protein abundance may range from 10 copies/cell to 10,00,000/cell.  A typical proteomics workflow involves various steps such as protein isolation, digestion of proteins into peptides followed by further fractionation of peptides using various electrophoresis (one-dimensional and/or 2D gel electrophoresis) and chromatography techniques (size exclusion chromatography, ion-exchange chromatography, high performance liquid chromatography [LC] etc.) and then these fractionated peptide mixtures are subjected to MS and identified with the help of various databases. , e.g. From the identification of peptides and proteins, MS can be used for relative quantitation of peptides. Although mass spectrometers are inherently not quantitative, various methods have been used for relative quantitation of proteins. ,
Recent advances in MS-based proteomics have made it an essential tool for use in the clinical practice. The goal of clinical proteomics is the identification of disease-specific biomarkers. , A biomarker is a substance used as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic invention. , Blood plasma/serum is one of the best sources of biomarkers as it remains in contact with nearly all cells of the organism and is also easily accessible. However, several preanalytical variables need to be considered for successful identification of markers using plasma. 
Vitamin B 12 and holo-transcobalamin
Vitamin B 12 is a water-soluble vitamin and is a cofactor of two enzymes, methionine synthase catalyzing the conversion of homocysteine to methionine and methylmalonyl coenzyme a mutase catalyzing the conversion of L-methlmalonyl-CoA, to succinly-CoA. The conversion of homocysteine to methionine is important for the synthesis of S-adenosylmethionine, which is the only methyl donor in a cell and it is an important reaction driving one carbon metabolism. , This methyl group can be transferred to DNA, RNA or protein. On the other hand, methymalonyl-CoA to succinly-CoA conversion is important for degrading propionic acid, which is required for maintaining the fat metabolism homeostasis. The dietary deficiency of vitamin B 12 is known to cause anemia and various neurological disorders. ,, The nutritional deficiency of vitamin B 12 during pregnancy has already been reported to cause fetal loss, premature birth, and fetal growth retardation. , We have already shown that the vitamin B 12 deficiency is associated with coronary artery disease (CAD) in India.  Further, deficiency of vitamin B 12 leads to the elevated levels of thiol amino acids such as homocysteine and cysteine. Homocysteine is an independent risk factor for the development of CAD.  Recently, elevated cysteine levels have also been associated with metabolic diseases and obesity. 
Vitamin B 12 is absorbed in the intestine with the help of intrinsic factor. In the plasma, Vitamin B 12 is circulated bound to two proteins haptocorrin and transcobalamin (TC).  Vitamin B 12 is bound to haptacorrin is not available to the cells for absorption. However, only approximately 25-30% of the TC is bound to Vitamin B 12 and is available to cells in the active form.  Therefore, holo-TC (HoloTC) is considered as the active vitamin B 12 and is also considered as the ideal biomarker to detect early vitamin B 12 deficiency. Herbert for the first time suggested HoloTC as the first marker to be declined in the vitamin B 12 deficiency ,, and is also been confirmed in the vegetarian population.  Vitamin B 12 deficiency is common in Indian population.  Various studies suggest that around 40-70% of the Indian population is vitamin B 12 deficient.
Thus, it is important to understand the physiological changes caused by the low HoloTc levels. In this study, we attempt to delineate the global proteome changes that occur in healthy controls (males and females) with low and high levels of HoloTC using a quantitative proteomics approach.
| Material and Methods|| |
Dithiothreitol (DTT), iodoacetamide and formic acid were procured from Sigma (St. Louis, MO, USA). Trypsin (modified, sequencing grade, V511) was procured from Promega. The isobaric tags for relative and absolute quantitation (iTRAQ) reagents were procured from ABSciex. The LC-MS grade water and ACN were procured from J. T. Baker. The isotope-coded affinity tag (ICAT) strong cation exchange chromatography column was purchased from ABSciex, Multiple Affinity Removal Column (Hu-6, 4.6 mm × 50 mm) was procured from Agilent. All other chemicals used were of analytical grade.
Ethics approval and consent form
Ethics committee approval was obtained from CSIR-Institute of Genomics and Integrative Biology. A written consent form based on the principles of the Helsinki Declaration was obtained from every healthy donor.
Subjects and study design
Plasma samples of healthy males (n = 8) and females (n = 8) were taken and divided on the basis of HoloTC levels (>55 pmol/l and < 25pmol/l) as follows: Male with low HoloTC levels < 25pmol/l, Male with high HoloTC levels > 55 pmol/l, Female with low HoloTC levels < 25pmol/l, Female high HoloTC levels > 55 pmol/l.
Blood sampling and storage
Fasting blood plasma was collected in an EDTA vacutainer by venipuncture method and plasma was separated after centrifugation at 1300 × g at 4°C for 15 min. The collected plasma was aliquot (500 μl) into polypropylene tubes, stored at −80°C, and thawed immediately before the study. 
Holo-transcobalamin was measured by sandwich enzyme-linked immunosorbent assay (Axis-Shield Diagnostics Limited, Scotland, UK) following the manufacturers' protocol.
Sample preparation using multiple affinity immunodepletion
Individual plasma samples from each group were immunodepleted for six high abundant proteins included albumin, IgG, antitrypsin, IgA, transferrin and haptoglobin using Multiple Affinity Removal Column (Hu-6, 4.6 mm × 50 mm, Agilent). Prior to spin removal affinity chromatography, plasma samples were diluted using Buffer A according to manufacturer protocol. Any particulates from plasma were filtered using Millipore (0.22 μm spin filter, Bedford, MA, USA). 200 μl of diluted plasma was loaded onto a spin cartridge. The flow-through fraction comprising low abundance proteins was collected between 2.50 min, which was further concentrated and were subjected to buffer exchange in with 0.5 M TEAB (pH 8.5) using 3 kDa Amicon Ultra centrifugal filter devices (Millipore, Billerica, MA, USA). The filter device was spun at 7000 × g for 20 min at 10°C.
Protein assay, protein reduction, and alkylation
The total protein concentration of low abundance proteins collected from the immunodepletion column flow-through of each subject group was determined using Bradford reagent (Sigma USA). Samples were then reduced with 25 mM DTT for 30 min at 60°C and alkylated with 55 mM iodoacetamide at room temperature for 15-20 min to irreversibly block free cysteine groups.
Protein digestion and isobaric tags for relative and absolute quantitation labeling
After reduction and alkylation, 50 μg of protein from each sample were digested overnight for 16 h with trypsin in 1:10 ratio at 37°C. Samples were then labeled with iTRAQ reagents following the protocol provided by the manufacturer (AB Sciex, Foster City, CA, USA). In brief all vials of iTRAQ labeling tags 113, 114, 115, 116, 117, 118, 119, 121 were reconstituted in absolute isopropanol (50 μL).  The entire contents of each iTRAQ vial were added to each sample according to the group and incubated for 2 h at room temperature, and the reaction was quenched by 50 μL LC-MS grade milli-q water. The 8 iTRAQ labeled samples in each set were then pooled separately into a single vial and vacuum dried using vacuum concentrator (Eppendorf, USA). The dried sample were then reconstituted in 8 mM Ammonium formate buffer pH 3 and is subjected to further cation exchange fractionation via ICAT Cartridge (AB Sciex, Foster City, CA, USA). The peptides were than eluted with 500 μL of increasing concentration of Ammonium formate buffer pH 3 from 35 mM, 75 mM, 100 mM, 125 mM, 150 mM, 250 mM, 350 mM, 500 mM and vacuum dried
Reverse phase separation and mass spectrometry
The peptide fractions were loaded onto reverse phase C18 analytical column (ChromXP nanoLC column 75 μm × 15 cm, 3 μm 120 Å) associated with trap column (ChromXP nanoLC Trap column 350 μm × 0.5 mm, 3 μm 120 Å). The peptide separation is performed using Eksigent nano-LC (Ultra 2D) coupled with 5600 triple time-of-flight (TOF) (AB Sciex). The peptide fractions were premixed in loading buffer mobile phase A (95% water, 5% and 0.1% formic acid) and 10 μL was loaded on a trap column with a flow rate of 10 μL/min. The retained peptides were washed isocratically by loading buffer for 40 min to remove excess salt. The peptides were then resolved on an analytical column with a multistep linear gradient of loading buffer mobile phase A (95% water, 5% and 0.1% formic acid)) and elution buffer mobile phase B (95% ACN, 5% water and 0.1% formic acid) at a flow rate of 250 nL/min. The gradient started at 5% buffer B and was held for 1 min, with linear increases up to 30% B at 80 min and 90% B in another 20 min. The gradient was held at 90% B for 5 min before being re-equilibrated to 5% B for 15 min. The triple TOF 5600 (AB Sciex) was operated in information-dependent acquisition (IDA) mode. The full MS spectra were acquired in positive ion mode in m/z 350-1200 Da with a 0.25 s TOF MS accumulation time, whereas the MS/MS product ion scan was performed in the mass range of 100-2000 Da with a 0.1 s accumulation time. The MS settings were as follows: Ionspray voltage floating = 1950 V, curtain gas = 30, ion source gas 1 = 20, interface heater temperature = 130, and declustering potential = 80 V. For 24 s former target ions were excluded and 20 candidate ions were monitored per MS cycle. IDA advanced "rolling collision energy" were applied for subsequent MS and MS/MS scans.
Database search and analysis
Data in the constituted MS and MS/MS spectra scan was obtained from Triple TOF 5600 in the form of.wiff files. These.wiff files from each iTRAQ experiment were submitted for protein identification to ProteinPilot™ software (v. 4.5 Applied Biosystems/MDS Sciex, Foster City, CA). using a Paragon search method against the homo sapiens SwissProt database. The search parameters were given as follows: trypsin as the digestion enzyme with two missed cleavages, IAA modification on cysteine residue, iTRAQ 8-plex modification of the N termini of peptides and of the side chains of lysine, bias correction was applied, and proteins were identified with global protein false discovery rate (FDR) of 1%.
| Results|| |
With powerful MS tools massive amounts of data can be generated, but this massive data needs to be interpreted logically. Interpreting data efficiently require robost bioinformatics tools. ProteinPilot™ Software (Protein Pilot v4.0) from AB SCIEX provides such a platform to identify and quantify proteins for discovery and characterization. For protein identification MS/MS fragmentation data in Protein Pilot™ Software are processed via firstly preprocessing of raw data into a simplified peak lists, averaging of similar spectra and filtering of good quality spectra. After preprocessing, the peptide hypotheses is made which is the based on de novo sequencing of sequence tags and database search approach. The best peptide hypotheses are then scored using feature probabilities and then with the help of the Pro Group™ Algorithm proteins are inferred by considering the portions of sequences in proteins for which there is observed evidence of spectra. This algorithm is different from BLAST, which analyse the similarity between sequences in a database, irrespective of experimental data. The proteins inferred were then subjected to FDR analysis using decoy database searching. 
In order to study the differential proteomics profile of individuals with Low and High HoloTC levels, we performed iTRAQ-based proteomics experiments to identify proteins that are differentially expressed in 8 controls with low HoloTC and 8 individuals with high HoloTC. The general and clinical characteristics of the study population have been shown in [Table 1]. In the two replicate designs of 8-plex iTRAQ experiments [Table 2]a, we identified 108 and 102 proteins with 1% protein global FDR respectively using Protein Pilot v4.0. The summarized results of two iTRAQ experiments are shown in [Table 2]b. The proteins were considered differentially expressed if the ratio was > 1.2-fold or < 0.8-fold in any three of the four groups with same trend or differential expression in at least two groups albeit no change in other two. Of these proteins, 22 proteins (11 differentially expressed in males and 11 in females) were found to differentially expressed among males and females during vitamin B 12 deficiency [Table 3]a and b. Interestingly, among males and females 7 proteins were up-regulated whereas only 4 were found to be down-regulated. Only two proteins, apolipoprotein B and alpha-2-HS-glycoprotein were common among males and females with low Holo-TC level. apolipoprotein B showed up-regulation in low Holo-TC subjects in both males and females. However, alpha-2-HS-glycoprotein was downregulated in males and up-regulated in females in low Holo-TC subjects.
Annotation of dysregulated proteins: GeneCodis 2.0
To identify the pathways that were enriched with the differentially expressed proteins in high and low HoloTC individuals, functional analysis were performed using GeneCodis. , In the GeneCodis algorithm, a list of genes as input is given and with respect to a reference list biological annotations are inferred. GeneCodis uses reference set, as all genes from the given reference genome chosen from NCBI Entrez Gene database.  Annotations from several sources such as GO and KEGG etc., are assigned to genes in the input list. The apriori algorithm is then applied to locate sets of annotations that are co-occurring in the input list frequently followed by statistical testing and P value adjustment. The GO biological process analysis revealed changes in important biological pathways including lipid metabolic process, blood coagulation, cholesterol metabolic process, transport and lipoprotein metabolic process [Figure 1]a and b. KEGG pathway analysis indicated that four significant pathways involving complement and coagulation cascade, vitamin digestion and absorption, Staphylococcus aureus infection, fat digestion and absorption were altered [Figure 2]a and b.
| Discussion|| |
Holo-transcobalamin is considered as the early marker of the vitamin B 12 deficiency. In this study, we performed the global proteomics study of the control subjects with low and high HoloTC levels and catalogued differentially expressed proteins. The global proteomics study revealed modulation of lipid metabolic process, blood coagulation, cholesterol metabolic process, and transport and lipoprotein metabolic process. Thus, alteration in lipid metabolism could be a major outcome during vitamin B 12 deficiency. We have already shown that vitamin B 12 deficiency is a contributing risk factor in the progress of CAD among the strict vegetarian population in India.  In this study, we found that both among males and females the levels of apolipoprotein B were up-regulated among group with low HoloTc levels. apolipoprotein B is a known risk factor for cardiovascular disease. ,, Thus, lower HoloTc levels could alter apolipoprotein B, which may predispose individuals to risk of CAD. Further, the levels of apolipoprotein F (apoF) were found to be higher in males with low HoloTC when compared to males with high HoloTC. apoF is a secreted sialoglycoprotein that exist in the high-density lipoprotein (HDL) and low-density lipoprotein (LDL) fraction of plasma. ApoF is also known as lipid transfer inhibitor protein (LTIP) and it restrains cholesteryl ester transfer protein (CETP)-mediated transfer events between lipoproteins. Lagor et al. overexpressed apoF in mice and had shown the reduction in HDL cholesterol levels by increasing the macrophage cholesterol efflux and increased clearance of HDL from the circulation.  Paromov and Morton suggests that the LTIP is a regulator of HDL metabolism mediated by CETP.  Lagor et al. has also generated the ApoF/LDLR (low-density lipoprotein receptor) double knockout mice and has shown the reduction in the development of atherosclerotic lesion via the reduced expression of Stat2, which is a critical player in Type I interferon (Type I IFN) pathway. He proposed that STAT2 had an important role in atherosclerosis via the Type I IFN pathway.  Thus, increased Apo F level could be another important player in increasing risk to CVD contributed by low HoloTC levels. An apolipoprotein C-I level was upregulated in males with low HoloTC when compared to males with high HoloTC. Apolipoprotein C-I levels are known to be associated with CAD.  Björkegren et al. has shown the accumulation of apolipoprotein C-I bound to very low LDL in normolipidemic patients with CAD.  Thus, low HoloTC levels marked by the vitamin B 12 deficiency lead to altered lipid metabolism due to altered lipoprotein profiles. It has been already shown that the vitamin B 12 deficiency adversely affects lipid profile in the Indian population. 
Interestingly, we found the levels of the fibrinogen gamma chain to be upregulated in males with low HoloTC when compared to males with high HoloTC. It is evident that the higher levels of fibrinogen are associated with the risk of CADs. ,,, Mannila et al. has shown that the elevated plasma levels of fibrinogen gamma is associated with the risk of myocardial infarction, which was further confirmed by the presence of polymorphism in two alleles FGG 9340T and FGA 2224G determined in Stockholm Coronary Artery Risk Factor study.  Cheung et al. and Lovely et al. have shown that the elevated fibrinogen gamma ratio leads to CAD progression. , Fibrin, in general, is found to be elevated in atherosclerotic lesions. Fibrinogen and its metabolites formed after its decomposition attracts various adhesion molecules in the endothelium triggering collagen synthesis.  It also enhances the vascular permeability leading to leukocytes attraction deposition in the endothelium.  In progressive atherosclerotic plaques, fibrin contributes to the formation of lipid nucleus formed by the close interaction of fibrin with LDL. , Moreover, Naruszewicz et al. has shown that the higher fibrinogen levels can be corrected by giving vitamin B 12 in the patients suffering from Ischemic heart disease. 
Alpha-l-acid glycoprotein (AAG), which is known to be a major drug binding protein present in plasma like albumin was found to be down-regulated in males with low HoloTC levels as compared to males with high HoloTC levels. Alteration in its levels can have major clinical consequences. AAG is a naturally occurring anti-inflammatory molecule and is known to increase in response to inflammation. However, Snyder and Coodley et al. has shown the inhibition of platelet aggregation by AAG , and biological function of the AAG is largely unknown. Inflammation is not proven to cause heart disease, but it is commonly seen in heart disease patients. The reduced levels of AAG can be attributed to decreased ability to prevent platelets aggregation and inflammation in CVD.
Among females with low HoloTC as compared to High HoloTC, Angiotensinogen (Serine or cysteine) proteinase inhibitor was found to be up regulated. Angiotensiongen is cleaved by renin to form angiotensin, which is then cleaved to the angiotensin II by angiotensin converting enzyme. The angiotensin II is a physiologically active enzyme involved in maintaining hemodynamic pressure in the body. Various studies show that the angiotensin II plays a critical role in mechanical destabilization of the atherosclerotic plaque by causing vasoconstriction. , Tewksbury and Dart have shown the increased expression of a low molecular weight form of angiotensinogen in hypertensive pregnant females.  Thus, it could play a role in the early progression of CAD marked by low HoloTC levels.
Alpha-1-antichymotrypsin (AACT) levels were found elevated in this study. It is an acute phase protein produced during inflammation. Tachikawa et al. has shown that the frequency of AACT variant A1252G was higher in within patients with ischemic CVD.  Further Lok et al. has also shown that the levels of AACT levels are higher in patients suffering from chronic heart failure.  The levels of serum amyloid P component (SAP) were also found to be elevated in females with low HoloTC levels as compared to females with high HoloTC levels. It is amyloid P component found in serum and belongs to a family acute phase inflammation protein. However, not much is known about the function of this protein though some studies suggest that the elevated levels of SAP is indicative a heart disease.  Further the levels of thyroxine-binding globulin were found to be low in females with low HoloTC levels when compared to females with high HoloTC levels. Thyroxine-binding globulin is one among the three other thyroid-binding proteins found in blood plasma. The decreased levels are implicated in hypothyroidism, which in turn leads to decrease in sex hormone-binding globulin levels.  Decreased SHBG is already known to be a risk factor for the development of cardiovascular disease in women. 
| Conclusion|| |
Our study emphasizes on the importance of vitamin B 12 metabolism and monitoring the levels of HoloTC. Taken together, this study reveals major perturbations in the lipid metabolism; major players being apolipoprotein B and F and proteins involved in inflammatory responses. These alterations suggest that the low HoloTc levels can be seen as a risk in the development of CAD. Further studies are needed for better understanding of the physiological alteration underlying the low HoloTC levels.
| Acknowledgments|| |
We acknowledge the financial assistance from Council of Scientific and Industrial Research (CSIR), Ministry of Science and Technology, Government of India, India under the XII FYP project titled "Centre for Cardiovascular and Metabolic Disease Research (BSC0122)." SV acknowledge the junior research fellowship from UGC. TB acknowledges the senior research fellowship from CSIR.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]