Mass spectrometry facility at AMRF is equipped with
- ESI -MS/MS (OrbitrapVelos Pro) with two sources
- Nano Spray Source
- Easy Spray Source
Description of the Equipments
Both the equipments have a nano-RPLC which can be operated at a pressure between 300 to 1000 bar. The nano-RPLC has a two-column set up, pre-column and analytical column. Samples are first loaded to a pre-column (Acclaim PepMap 100 C18, 3 m particle size, 100, 75 m2 cm) and peptides are separated prior to MS/MS using an analytical column 15 or 50 cm (Acclaim PepMap C18, 3 m particle size, 100 , 75 m x 15 cm /50 cm) column.
For reducing the complexity of the samples two optimized work flows are developed at our laboratory:
- Protein pre-fractionation using SDS-PAGE
- Peptide pre-fractionation using IEF
- Protein pre-fractionation using glycoprotein enrichment
- Glycopeptide enrichment is under development
Protein Identification using pre-fractionation with HCET cell lines
|Sample name||Proteins identified by insolution digestion followed by peptide IEF fractionation and mass spectrometry||Proteins identified by prefractionation on SDS-PAGE followed by In-gel digestion and mass spectrometry|
|HCET Cell lines Cytosolic fractions||2861||2913|
Method optimizations for separation of peptides in LC:
The potential of LC-MS/MS depends upon HPLC separation of peptides through the Reverse Phase chromatography column. Hence the gradient with varying components of Sol A (0.1% Formic acid with 5% ACN ) and Sol B (95% ACN with 0.1% Formic acid) has to be optimized over a time period of 60 min to 240 min depending on the retention time of the peptides which inturn is dependent on the complexity of the peptide mixtures.
Protein identifications using LC-MS/MS approach are carried out comprehensively with various ocular samples with an objective of creating occular protein database of Indian population. Snapshot of the various samples used for optimizations and data generation for protein identifications is given in the table below: Total run time varies from 60, 90, 130, 240 min/run/ injection depending on the sample complexity. Currently, we perform Protein identification and Relative quantification (using Label free methods).
Details of complex samples which has been analysed at our MS facility:
|Sample||No. of runs||Total proteins identified|
|HeLa Cell lines||>60 ||2393|
|HCET cell lines||>50||4809|
Proteomics software currently available at our facility
Proteome Discoverer Software
Thermo Scientific Proteome Discoverer Software is a flexible, expandable platform for the analysis of qualitative and quantitative proteomics data.
PEAKS Studio Software
PEAKS Studio Software is for de novo sequencing; produced by Bioinformatics Solutions of Waterloo, Canada.
MASCOT Server Software
Mascot is a powerful search engine which uses mass spectrometry data to identify proteins from primary sequence databases
SIEVE Software for Differential Analysis
Confidently and reproducibly identify components with statistically significant intersample differences in abundance using Thermo Scientific SIEVE software.
Speed characterization of complex glycans; use SimGlycan software to interpret the rich MSndata produced by Thermo Scientific mass spectrometers
ProSightPC 2.0 Software
The first stand-alone software for analyzing top-down proteomics data, has been enhanced to add support for middle-down and bottom-up experiments.
Protein Deconvolution Software
Significantly improves the identification and characterization of intact proteins from mass spectrometric data.
A flexible Windows-based data system that provides instrument control and data analysis for the entire family of mass spectrometers and related instruments.
Two well trained technicians, Ms Sujitha and MsKalaiselvi are on job at the MS a facility to address the day to day operations, routine Maintainance of MS and sample resuspension and sample loading in LC.
Human Organ Culture Anterior Segment (HOCAS)
Use of facility: Human Organ culture Anterior Segment (HOCAS) is an ex vivo model system for screening drugs with TM outflow modulating property using human donor eyes
Shimadzu High Pressure Liquid Chromatography (HPLC)
Use of facility: The analysis of drugs, vitamins and or chemical substances in biological fluids are being carried out using this facility.
LEICA Confocal Microscope SP2
Use of Facility: The Leica TCS SP2 AOBS is a programmable system free of excitation and emission filters. The filterless detector allows defining wavelength bands for each channel, allowing the user to maximize emission sensitivity and separate closely spaced fluorescent dyes. The Leica SP2 AOBS confocal microscope is equipped with a 3-laser configuration (488/561/633nm) and can be used for a variety of biological samples. This instrument provides high-resolution images using a variable pinhole for optical z sectioning and laser scanning.
Usage: It is used for characterizing immunostained tissues/tissue sections/cytospin preparation of cells more specifically for (i) identifying and quantifying limbal epithelial stem cells by two parameter analysis - high expression of nuclear transcription factor -p63 with high nucleus to cytoplasmic ratio; (ii) characterizing the limbal stromal niche - cellular and extracellular matrix components and (iii) to identify the tissue macrophage subpopulation in retinoblastoma.
Zeiss Palm Beam Laser capture micro dissection microscope
Use of facility: This system enables laser assisted isolation of individual /group of cells from fixed sections that can be used further for genomics/proteomics. It can also be used to separate cells of interest from live culture for subculturing. The laser used for microdissection is 355 nm pulsed-laser which does not affect the quality of DNA/RNA/protein and hence the downstream processing.
Usage: This facility is currently used to enrich the corneal epithelial stem cells from a heterogenous population, in order to understand the gene expression patterns and signalling mechanisms specific for stem cells. It is possible to remove the unwanted cells which may alter our results and conceal the signals of the relevant cells.
Good Manufacturing Practice (GMP) facility
The GMP facility for culturing LESCs/BMESCs has been established at Aravind Medical Research Laboratory in 2009. The basic infrastructure - Class 1000 facility for culturing LESCs and BMESCs in separate modules with the following equipments is available.
- The BioKlenz Bio-containment work station is a Class II - Type A2 unit in which 30% of the HEPA filtered downward air is exhausted through an exhaust HEPA filter and 70% is recirculated. This ensures a negative pressure work zone.
- The special feature of the air-jacketed CO2 incubators in lab modules 1 and 2 is the high temperature Contracon (90?C), which is a moist heat decontamination process. High quality sensors are mounted directly within the chambers for precise environmental measurements.
- Inverted phase contrast microscopes for observing the cells in culture.
- There is also a refrigerated centrifuge and a stirred water bath in common space, -30?C freezer and a refrigerator in the cold storage room for storing the media and other requirements for cell culture work.
- KlenzPortrecirculatory pass box is designed for material transfer between areas under different classification. This system is equipped with two SS doors with view panels of glass flush mounted, electro-magnetic interlocking arrangement and magnahelic gauge.
- A Sensaphone installed in the GMP facility, is to monitor the proper functioning of the air-jacketed CO2 incubators and -30C freezer. When there is some error in these instruments, then the alarm in the sensaphone conveys the message automatically to the concerned faculty through phone.
- In addition, air particle counting, air velocity and DOP tests have been done to ensure that the GMP facility meets the clean room criteria.
Use of facility: The BD FACSCalibur platform allows users to perform cell analysis that includes (i) immunophenotyping including intracellular cytokine production, (ii) quantification of cytokines (up to eight cytokines/assay) using cytometric bead arrays, (iii) rare event analysis wherein millions of cells are analyzed to achieve a statistically significant sampling of the subset of interest, (iv) DNA analysis that ensures that high-resolution DNA ploidy and S-phase determinations are easy to achieve.
Usage: The flow cytometer is used (i) to characterize the cultured limbal/corneal stromal cells for the expression of mesenchymal/embryonic stem cell markers and (ii) for identifying the cytokine (intracellular and secreted) profiles after in vitro stimulation of PBMCs/neutrophils with crude hyphal extracts to understand the immunopathogenesis of fungal keratitis .
ABI 7900 HT Fast Real Time PCR
Work Progress of the facility:
Real Time PCR has been installed in Department of Molecular Genetics in 2008. This machine has been used by Genetics, Microbiology and Ocular Pharmacology. This instrument is mainly used for the INDEYE study on genotyping Cataract and AMD samples by Allelic discrimination assay. The other research groups utilize this instrument for study of the gene expression, deletion/duplication analysis, copy number variations and also for species identification. So far more than six International research articles were published using the data generated from the instrument.
ABI 3130 Genetic Analyzer
Work Progress of the facility:
Genetic Analyzer has been installed in the Department of Molecular Genetics in 2007. Since then, it has been widely used by several groups of AMRF including Immunology, Microbiology and Proteomics. The sequencing instrument has been used for mutation screening, genotyping (Multi Locus Sequence Typing and Multiplex Ligation Dependent Probe Amplification), species/strain identification & confirmation and checking the clones. The data generated from sequencing in multiple research projects was presented in many national and international meetings and published in highly reputed journals of ophthalmology.
Name of the facility: Bioplex
- Bioplex reader
- Bio-Plex Manager 6.0 software, PC
- Vacuum station and manifold
- Microplate Shaker
Work/Progress of the facility:
Bio-plex allows for the simultaneous analysis of multiple analytes in small sample volumes including cytokines, total proteins, and phosphoproteins. The instrument was purchased and installed in Nov 2011. Along with the instrument, Pro-human cytokine 21 plex, Pro-human cytokine 27plex and BP pro human diab 10-plex kits (human diabetes biomarkers)were obtained and analysis was done in tear (fungal keratitis), aqueous humour (POAG), serum (DR) and Stem cell culture supernatants in 2012.
AMRF Biocomputing Center (ABC)
Work/Progress of the facility
The Biocomputing center provides a core computational facility to support eye-related research by developing and maintain computing facilities including data storage, database development and maintenance, algorithm development, analysis software tools, hardware support. Also it provides to customize data analysis tailored to the needs of individual research projects across all the research groups and extend this service to others on mutually acceptable terms. In addition, it helps to train manpower by way of workshops and hands on training.
Details of activities in progress:
- Data storage and retrieval:
- Providing network storage, data management and handling to all research groups at AMRF. A large amount of data is generated at the all level of research in various research groups such as Imaging, Microarray, NGS and Mass spectrometry. All these continuous generation of data files of large sizes that are to be archived appropriately as well as backed up on a regular basis.
- Method development:
- Developing a structure-based bioinformatics method to the Analysis of Single Nucleotide Variants (SNVs) and Prediction of its Association with Genetic Eye Diseases.
- Developing an exome analysis software pipeline to detect and filter clinical variants for genetic eye disorders using Next-generation sequencing clinical data
- Web Database and Hosting:
- Creating a complete mutational database of eye genes, which includes all the information of candidate genes, Insilco genotype-phenotypic correlation and Age-penetrated statistical methodology to distinguish the each mutation from disease causing variation.
- Data analysis:
Providing data analysis from different sources that require computers with powerful processing capabilities along with high memory requirements to carry out
- Comparative bacterial genome analysis using whole genome short-gun sequencing projects.
- Comparative transcriptomic analysis of fungal pathogens.
- RB1 Clinical variant analysis using next-generation sequencing data from Retinoblastoma patients
- Microarray data analysis for AMRF projects on Diabetic Retinopathy, Stem Cell research, Bacterial Keratitis and Retinoblastoma.