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3-D Structure of Human Serum Albumin

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Mark E. Bier
Director, CMA
mbier@andrew.cmu.edu
412-268-3540 (office)
412-268-6897 (fax)


Roberto R. Gil

Director, NMR Facilities

rgil@andrew.cmu.edu

412-268-4313(office)


Carnegie Mellon University
Department of Chemistry
Mellon Institute Bldg.
4400 Fifth Avenue
Pittsburgh, PA 15213-2683

 

Liquid Chromatography and LC/MS


The CMA has two LCs available for LC/MS:

Thermo Scientific EASY-nLC 1200 nanoLC

Thermo Scientific Vanquish Flex Binary UHPLC


General Usage Guidelines:

         All samples must be filtered and degassed prior to usings the LCs.

         Users must reserve time on the instrument calendar prior to use.

         For more information about mass spectrometry, see our useful links.


 

Thermo Scientific EASY-nLC 1200 Nanoflow Liquid Chromatograph

The The Thermo Scientific EASY-nLC 1200 is a nanoflow liquid chromatograph capable of precise gradient flow (water/acetonitrile only) from 20 nL/min to 2 L/min with pressures up to 1200 bar. For LC-MS the EASY-nLC 1200 is coupled to a Thermo Scientific LTQ-XL linear ion trap (LIT) mass spectrometer with nanoelectrospray ionization (nESI) capabilities. The LTQ-XL has MSn capabilities, which is a powerful analytical tool for use in structural analysis. This mass spectrometer can provide molecular weight and sequence information for small peptides (MW below 2000 Da) at the femtomole level with proper analyte introduction.

 

EASY-nLC 1200 USER'S MANUAL in PDF format. Please read prior to nLC-MS inquiries.


LTQ-XL Linear Iontrap MS USER'S MANUAL in PDF format. Please read for training.



NanoLC Overview:

         Pressure range: 0-1200 bar

         Flow range: 20-2000 nl/min (Recommended: 100-1000 nL/min)

         Samples injected via autosampler

         Software integration with MS and instrument control using Xcalibur 4.0

         NanoViper fitting technology enables low dead volume liquid flow path connectivity. Minimizes peak broadening due to mixing.

         nano-ESI ionization source developed in-house utilizes a 10 m emitter.


MS Overview:

         Mass Ranges: 15-200 Th, 50-2000 Th, 200-4000 Th

         Resolution: 0.05 FWHM with Ultra ZoomScan

         Sensitivity: 250 femtogram reserpine in 1:1 isopropanol:water at 400L/min flow rate for both MS/MS and MSn.

         ZoomScan for isotopic resolution across full-range scan

         Ultra ZoomScan for ultimate resolution

         Turbo Scan for ultra-fast scan to improve signal-to-noise and sampling rate

         Positive/negative ionization modes

         Multiple Ionization Strategies:

         Electrospray Ionization (ESI) -- (Thermo Scientific Ion Max Source)

         Nanoelectrospray Ionization (nESI) -- (Thermo Scientific Nanospray Flex Source)

         Direct Analysis in Real Time (DART) -- (IonSense DART-OS Source)

         Pulsed Q Collision Induced Dissociation (PQD) enables trapping of low mass fragment ions.

         High Resolution Isolation allows for the separation of an isobaric interfering species down to 0.3 Da or for isolation of thermally labile compounds.

         Automatic Gain Control (AGC) used to ensure ideal number of ions stored in linear ion trap each scan.

         MSn for n=1 to 15 for structural analysis

         Advanced Data Dependent Experiments possible (i.e. Triple Play, Neutral Loss, Ion Tree)

         Linear ion trap with radial ejection. Dual conversion dynode detectors used to capture high capacity of the ion signal.

         Automated system calibration performed by CMA staff only.


Sample Preparation:

         Filtered

         Degassed


 

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Thermo Scientific Vanquish Flex Binary UHPLC

The The Thermo Scientific Vanquish Flex Binary UHPLC is a liquid chromatograph capable of precise, binary gradient flow from 1 L/min to 8 mL/min with pressures up to 15,000 psi. For LC-MS, the Vanquish Flex Binary UHPLC is coupled to a Thermo Scientific Exactive Plus EMR Orbitrap mass spectrometer with nanoelectrospray (nESI), electrospray (ESI), or direct analysis in real time (DART) ionization capabilities. The Exactive Plus EMR has high resolution and accurate mass (HRAM) capabilities for high confidence molecular weight determinations. This mass spectrometer can provide molecular weight and sequence information for small molecules at the femtomole level with proper analyte introduction. Additionally, the Exactive Plus EMR provides extended mass range (EMR) capabilities up to m/z 20,000 for the analysis of large macromolecules (e.g. nanoparticles, protein/protein complexes, polymers).

 

Vanquish Flex Binary UHPLC Product Brochure in PDF format. Please read prior to LC inquiries.


Vanquish Flex Binary UHPLC Specification Document in PDF format. Please read prior to LC inquiries.



UHPLC Overview:

         Pressure range: 290-15,000 psi

         Flow range: 0.001-8 ml/min

         Samples injected via autosampler

         Software integration with MS and instrument control using Xcalibur 4.0

         NanoViper fitting technology enables low dead volume liquid flow path connectivity. Minimizes peak broadening due to mixing.


MS Overview:

         Mass Ranges: 50-6,000 Th (standard mode), 350-20,000 Th (EMR mode)

         Resolution: Up to 140,000 (FWHM) @ m/z 200

         Mass Accuracy:

         Standard mode: <1 ppm RMS (internal); <3 ppm RMS (external)

         EMR mode (external): <3 ppm RMS (350-6,000 Th); <5 ppm RMS (6,001-10,000 Th); <10 ppm RMS (10,001-15,000 Th)

         Sensitivity: 500 fg buspirone on-column S/N 100:1 acquired with H-ESI II probe

         Max scan rate: Up to 12Hz at mass resolution setting of 17,500 @ m/z 200

         Positive/negative ionization modes

         All-ion-fragmentation (AIF) with either:

         In-source collision-induced dissociation (CID)

         Higher-energy collisional dissociation (HCD)

         Multiple Ionization Strategies:

         Electrospray Ionization (ESI) -- (Thermo Scientific Ion Max Source)

         Nanoelectrospray Ionization (nESI) -- (Thermo Scientific Nanospray Flex Source)

         Direct Analysis in Real Time (DART) -- (IonSense DART-OS Source)

         Automated system calibration performed by CMA staff only


Sample Preparation:

         Filtered

         Degassed


 

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