Mass Spectrometry Facility provides services for identification and quantification of small and large molecules like secondary metabolites, peptides and proteins. The facility houses ESI-MS: TripleTOF^® 5600+; MALDI TOF/ TOF Mass Spectrometer: SCIEX TOF/TOF^TM 5800; ESI-Mass Spectrometer: SCIEX QTRAP^® LCMS/MS 6500+ and Perkin Elmer Flexar^TM HPLC system.

1. ESI-Mass Spectrometer: SCIEX TripleTOF^® 5600+

Mass Spectrometry enables determination of the molecular masses of small molecules as well as of large biomolecules such as proteins and peptides. The SCIEX Triple TOF 5600+ mass spectrometer is a high-resolution LC-MS/MS mass spectrometer with high sensitivity and high mass accuracy. The Triple TOF^® 5600+ has both ESI and APPI ionization sources and is hyphenated with an ExionLC for LC-MS analysis of small molecules and also with an Eksigent ekspertnanoLC 425 for qualitative and quantitative proteomics analysis.

2. MALDI TOF/ TOF Mass Spectrometer: SCIEX TOF/TOF^TM 5800

The facility also houses SCIEX TOF/TOF^TM 5800 mass spectrometer with EKSpot MALDI spotter for qualitative and quantitative proteomics analysis. The system is widely used for identification of protein/peptide, characterization of PTM, de novo sequencing and relative quantification using iTRAQ, SILAC, TMT etc.

3. ESI-Mass Spectrometer: SCIEX QTRAP^® LCMS/MS 6500+

The facility is equipped with SCIEX QTRAP^® LCMS/MS 6500+ system. It is the fastest and most sensitive QTRAP. It provides the best LOD's and LOQs to enable the detection and quantification of the widest scope of chemical compounds in the most challenging matrices. Improved polarity switching and MRM3 speeds allow faster chromatography and better throughput. The built in QTRAP functionality enables quantitative MRMs and qualitative QTRAP scans in the same injection to maximize throughput. Best being utilized for performing applications requiring ultimate sensitivity for ultra-low-level identification and quantitation of both small and large molecules and for confirmatory analysis of metabolites.

4. Perkin Elmer Flexar^TM HPLC system

The facility provides services for cation exchange/high pH fractionation of complex proteomics samples using Perkin Elmer Flexar^TM HPLC system which is coupled with automated fraction collector for wide coverage of proteome.

This facility hosts following state-of-the-art instruments: Super Resolution Microscope; Laser Scanning Confocal Microscope and High Content Imaging System.

1. Super Resolution Microscope: Elyra PS1, Carl Zeiss

Structured illumination microscope is a widefield technique in which a grid pattern is generated through interference of diffraction orders and superimposed on the specimen while capturing images. The grid pattern is shifted or rotated in steps between the capture of each image set. Following processing with a specialized algorithm, high-frequency information can be extracted from the raw data to produce a reconstructed image having a lateral resolution approximately twice that of diffraction-limited instruments and an axial resolution upto 120 nanometers.

About Microscope: The Microscope is framed in an inverted fluorescence research microscope (Axio observer 7) with motorized stage. There are other super resolution modules available with the system which are as follows:

• Total Internal Reflection Fluorescence Microscopy (TIRF)
• Photo Activation Localization Microscopy (PALM with 3D)
• Stochastic Optical Reconstruction Imaging (STORM)

It has a metal halide illuminator HXP120 for sample observation and 4 diode lasers to make imaging comprehensive. The lasers are:

• 405 nm Laser 50 mW
• 488 nm laser 100 mW
• 561 nm laser 100 mW
• 640 nm laser 150 mW

It is equipped with varied range of microscope objectives:

• 20X Plan-Apochromat (NA0.80)
• 63X C- Apochromat Water immersion (NA 1.2)
• 63X Plan-Apochromat Oil immersion (NA 1.40)
• 100X alpha Plan-Apochromat Oil immersion (TIRF Objective) (NA 1.46)

The system uses two different cameras for imaging:

• Andor EM CCD camera iXon DU897 (For TIRF, PALM and STORM imaging)
• Camera pco.edge sCMOS Version 4.2 (for SIM imaging)

Applications: System is used to find super resolved images (resolution up to 120 nm) of cells and their network filaments/fibers, organelles.

2 Laser Scanning Confocal Microscope: LSM 880, Carl Zeiss

The coherent light (excitation laser) passes through a pinhole aperture that is situated in a conjugate plane (confocal) with a scanning point on the specimen and a second pinhole aperture positioned in front of the detector (a photomultiplier tube). As the laser is reflected by a dichromatic mirror and scanned across the specimen in a defined focal plane, the emission from points on the specimen (in the same focal plane) pass back through the dichromatic mirror and are focused as a confocal point at the detector pinhole aperture. Due to this aperture, the extraneous light (out of focus light) is not detected by the detector thereby resulting in sharp images.

About Microscope: The Microscope consists of an inverted fluorescence research microscope (Axio observer 7) with following objectives:

• 10X Plan Apochromat (NA 0.45)
• 40X Plan-Apochromat Oil immersion DIC (NA 1.30)
• 63X Plan-Apochromat Oil immersion DIC (NA 1.40)

Observation and imaging units: A metal halide illuminator (HXP120) and HAL100 illuminator for sample observation and 5 lasers including Multi Argon lines for confocal imaging.

1. Diode laser 405 nm
2. Laser Argon Multiline 458/488/514 nm
3. Laser HeNe 543 nm
4. Laser HeNe 594 nm
5. Laser 633nm

Detection unit:

1. Transmitted light detector T-PMT
2. LSM 880 confocal scan head with 2MA-PMT and 1 GaAsP (for spectral detection)
3. High resolution digital microscopy camera AxioCam 503 mono

Applications: Fluorescence imaging of tissues, cells, live imaging and spectral imaging and dynamics experiments such as FRAP and FRET.

3. Micro Confocal High Content Imaging System: ImageXpress, Molecular Devices

The ImageXpress system is an integrated cellular imaging and analysis system that is designed for rapid, automated screening of fluorescently labelled fixed and live cells in microplates. It also provides environmental control for live cell imaging, moves fluids to and from cells for compound addition. The ImageXpress Confocal system is a high-content imaging system that can switch between widefield imaging and confocal imaging. The rapid autofocus and precision sample movement features of the microscope allow large numbers of high-resolution images to be acquired in the shortest possible time. All key optical and mechanical elements are motorized, with asynchronous command execution, allowing complete real-time control of the instrument configuration through the MetaXpress high-content image acquisition and analysis software. When used in combination with the powerful image analysis capabilities of the MetaXpress software, the instrument becomes an extremely flexible and programmable device, ideally suited for user-defined, high-speed automated assays.

Instrument Specifications

• ImageXpress Micro Confocal System is a fully automated confocal microscope with pinhole sizes of 50 µm and 60 µm.
• It is capable of taking images from a microplate (96 well/ 384 well / 1536 well) or from a microscopic slide.
• It is capable of detecting and imaging multiple fluorescent signals simultaneously.
• Transmitted light imaging can be done for non-fluorescent cells.
• Z-stack images can be acquired and analysed.
• Environmental control- multi-day, live cell time-lapse imaging under appropriate temperature, humidity and atmospheric conditions can be performed.
• This system also contains single-channel, fluidics robot. This robot picks up tips from a tip box and draws fluid from and delivers fluid to a given well on the compound and sample plates. The robot is capable of dispensing volumes between 3 μL and 200 μL.
• This system also has a hotel shelf with capacity for 88 standard microplates, where plates can be stacked for imaging. With the help of Sample Handling Robotic Arm, the plates can be removed from hotel shelf and can be loaded onto ImageXpress Micro Confocal System where images are acquired, and then returned to a home location.
• This entire system has been integrated with Cytomat 2C automated incubator through a Sample Handling Robotic Arm so that multiple plates can be scanned for time-lapse live cell imaging. This automated incubator has a capacity for 42 standard microplates.
• Automated barcode reader is also a part of this system.

Light Source

The external light source for the ImageXpress Micro Confocal System is a solid-state light source that has a rated lifetime of more than 20,000 hours. It has light source for excitation of DAPI, FITC, TEXAS RED, Cy3, Cy5 wavelengths.

Objectives

• 4X Plan Apo λ NA 0.20
• 10X Plan Apo NA 0.45
• 20X S Plan Fluor ELWD NA 0.45
• 40X S Plan Fluor ELWD NA 0.60
• 60X S Plan Fluor ELWD NA 0.70
• 100X CFI L Plan EPI NA 0.85

Applications

Application modules are the foundation of the MetaXpress Software analysis workflow. These modules provide automated image analysis for biology-specific applications, interactive image segmentation, and data results, and options for data recording and management. Currently, Our MetaXpress software has the following modules:

• Angiogenesis Tube Formation
• Cell Cycle
• Cell Health
• Cell Proliferation
• Cell Scoring
• Live Dead
• Micronuclei
• Multi Wavelength Scoring
• Translocation
• Neurite Outgrowth

This facility provides the platform for purification of proteins or biomolecules using FPLC systems. ÄKTA^TM pure M and ÄKTA^TMprime are available in the facility which will aid users in purification of proteins at preparative and analytical grades. This facility also provides the platform for measuring the biomolecular interactions using Biolayer interferometry and MicroScale Thermophoresis.

1. Bioreactor: New Brunswick BioFlo 415 SIP (7L & 14 L)

The BioFlo 415 is a versatile sterilization-in-place bioreactor that provides a fully equipped system in one compact package. It can be employed for batch or continuous culture with process control of pH, dissolved oxygen (DO), agitation, temperature, pump feed, anti-foam, level and addition analog/digital inputs and outputs.

2. Bio-Layer Interferometry (BLI): Octet RED96

Bio-Layer Interferometry (BLI) is a label-free technology for measuring biomolecular interactions. It is an optical analytical technique that analyzes the interference pattern of white light reflected from two surfaces: a layer of immobilized protein on the biosensor tip and an internal reference layer. Any change in the number of molecules bound to the biosensor tip causes a shift in the interference pattern that can be measured in real-time. The binding between a ligand immobilized on the biosensor tip surface and an analyte in solution produces an increase in optical thickness at the biosensor tip, which results in a wavelength shift, Δλ. Unbound molecules, changes in the refractive index of the surrounding medium or changes in flow rate do not affect the interference pattern. This is a unique characteristic of BLI and extends its capability to perform in crude samples used in applications for protein:protein binding, quantitation, affinity and kinetics.

Assays can now be performed over a wider temperature range of 15-40°C on the Octet RED96 system, allowing for kinetics measurements of unstable proteins at lower temperatures or biologically relevant molecules at physiological temperatures. An evaporation cover for microplates also allows for greater walk-away run time, with minimal sample evaporation for up to 12 hours. The new Octet Data Analysis HT software version released with the Octet RED96 system brings custom reporting options and multi-experiment analysis that reduces analysis time from hours to minutes.

3. FPLC: ÄKTA^TM pure M

ÄKTA pure M is a flexible and intuitive chromatography system for fast purification of proteins, peptides and nucleic acids from microgram levels to tens of grams of target product. ÄKTA pure M is a reliable system where hardware and UNICORN^TM software are designed to work together with columns and chromatography resins to meet purification challenges.

Depending on the column used with ÄKTA pure M, it can be used for the protein purification on the basis of charge (ion-exchange chromatography using columns: HiTrap Q, HiTrap SP etc.), affinity tags (affinity chromatography using columns: HisTrap, GSTrap, MBPTrap etc.) or size (size-exclusion chromatography using columns: HiLoad 16/600 Superdex 200 pg, etc.).

4. Multi Angle Light Scattering (MALS): Viscotek SEC-MALS 20

The Viscotek SEC-MALS 20 is a multi angle light scattering detector that can be used to measure the absolute molecular weight of molecules. It simultaneously measures the intensity of scattering at each angle which, in conjunction with a concentration detector, enables the calculation of the absolute molecular weight of proteins, synthetic & natural polymers and also molecular size expressed as the radius of gyration, Rg. For proteins, SEC-MALS measures the absolute molecular weight independent of column retention volume in order to study oligomeric state and aggregation.

5. FPLC: ÄKTA^TMprime

ÄKTAprime is the simple version of ÄKTA pure M. It is used to detect or purify biomolecules such as proteins in a mixture by recording the absorbance at 260 or 280 nm. The purified components of the mixture are collected in separate test tubes using a fraction collector. Ion-exchange chromatography, affinity chromatography, desalting, buffer exchange can be performed using this system.

6. MicroScale Thermophoresis (MST): Monolith NT.115

Monolith NT.115 uses MicroScale Thermophoresis (MST) technology to quantify binding events. One partner is typically labeled with a fluorescent dye and becomes an extremely sensitive reporter for all binding interactions. Monolith is all about accurately determining binding affinity in minutes. The strength of the interactions between a fluorescently labeled or intrinsically fluorescent sample and a binding partner (or ligand) are measured while a temperature gradient is applied over time. These events, weak or strong, cause a change in MST signal that is easily detected by plotting the normalized fluorescence against concentration of ligand. The results are automatically translated into precise and quantitative Kd values. Interactions can be studied in immobilization-free format.

7. Other services

The facility also provides services for the following instruments.

• Sorvall Lynx 6000 centrifuge, ThermoScientific
• FilterMax F5 Multi-Mode-Microplate-Reader, Molecular Devices
• Sonics vibra cell sonicator
• GEA Niro Soavi Panda Plus