Carnegie Mellon

  

 

 

3-D Structure of Human Serum Albumin

CMA Homepage
Information for users
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Mass Spectrometry
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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

 

Mass Spectrometry


The CMA has four mass spectrometers available for general use:

Thermo LTQ XL ESI/APCI Linear Ion Trap

Applied Biosystems Voyager DE-STR MALDI-TOF

Thermo LCQ ESI/APCI Ion Trap

Waters Q-TOF II ESI/APCI/Nanospray


General Usage Guidelines:

         Users expecting to have fewer than 3 samples per month, please use our sample drop-off service.

         Users expecting to run more than 3 samples/month for a year or longer should contact Dr. Mark Bier to schedule training.

         CMA provides a sample preparation bench for general use with resources such as pipettes, sample vials etc. but users must provide:

o        MALDI:  Calibrant, matrix, sample plate (Part # V700666 from Applied Biosystems).

o        LTQ-XL/LCQ/Q-TOF:  Two (2) square-tipped ("LC-style") gas-tight syringes. These are available from SGE, Inc. Removable-needle syringes are recommended, and be sure to select a syringe with "GT" in the code (for a gas-tight plunger).

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

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


 

Thermo Scientific LTQ-XL ESI/APCI Linear Ion Trap
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Thermo Scientific EASY-nLC 1200 Nanoflow Liquid Chromatograph

The Thermo Scientific LTQ-XL is a linear ion trap (LIT) mass spectrometer with electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) 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. Additionally, liquid seprations prior to MS detection can be made possible by coupling to the Thermo Scientific EASY-nLC 1200 liquid chromatograph with precise gradient flow (water/acetonitrile only) from 20 nL/min to 2 L/min with pressures up to 1200 bar.

 

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


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



System 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

         ESI and APCI sources

         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.


Calibration:

         Automated system calibration performed by CMA staff only


Sample Preparation:

         All samples should be in an ESI-friendly solvent system such as 50/50 Water/Acetonitrile, Water/Methanol, etc.

         For positive ion samples, a small amount of acetic acid (0.1% v/v) can be added to enhance the signal.  TFA is not recommended.

         For direct injection, prepare at least 100L of sample.  For flow-injection analysis 20L is typically sufficient.

         Recommended sample concentrations are as follow:

o        Small organics: 1-10M

o        Small peptides: 1-10M (pmol/L)

o        Large proteins: 2-10M (pmol/L)

o        Polymers: 10-100M


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.


 

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Applied Biosystems Voyager DE-STR MALDI-TOF

Funded by NSF grant #CHE-9808188

 

The Applied Biosystems (formerly PerSeptive Biosystems) Voyager STR MS is a matrix assisted laser desorption ionization (MALDI) time of flight (TOF) mass spectrometer. Ions are formed in the gas phase from a laser focused onto a matrix/analyte solid sample. The matrix compound is typically a small organic molecule, chosen for its ability to absorb photons at 337 nm from the nitrogen laser. Gas phase ions (positive ions are typically protonated) are then accelerated down the 2 meter flight tube with 20-25 keV of kinetic energy (KE). Low m/z ions hit the detector first followed by the slower, higher m/z ions. To a first order, the ions follow the equation KE=1/2mv2. Typical analyte molecules include polymers, peptides, proteins, and DNA.

See how MALDI works!

 

Voyager MALDI TOF MS USER'S MANUAL in PDF format. Read Chapters 1,3-5 for training.


Voyager MALDI TOF MS Quick Guide in PDF format. This guide has been created to guide users through a standard operation of the instrument. There are screenshots and illustrations to assist. It is suggested to use this document during your analyses.


System Overview:

         Mass range 1-400,000 Da

         Positive and negative ion modes

         Linear (R=3000) and reflector (R=15000) modes

         Post-source decay (PSD) with CID

         337nm nitrogen laser

         100 well standard sample plate (user-supplied)

         Polymeric analysis tool (PDI, Mn, Mw, Mz calculation)

         5ppm mass accuracy in reflectron mode with internal reference

         Femtomole-level detection with proper sample preparation


Calibration:

         Must be performed on a per-session basis by each individual user


Sample Preparation:

         Sample and matrix should be prepared in similar or miscible solvent systems.

         Preparation typically only requires 1L of sample

         Selecting the proper matrix has a large effect on results.  This decision can be based on previous successful MALDI analyses of similar samples, the NIST polymer recipes page for polymer samples, or see the general guide found in the Voyager manual (section 3.1.1 - "Selecting a Matrix").

o        Sinapic acid is the most common protein (>10kDa) matrix used.

o        α-cyano-4-hydroxycinnamic acid is the most common peptide (<10kDa) matrix used.

         Samples must be completely dry before loading into the instrument.

         For positive ion samples, a small amount of acetic acid or TFA (0.1% v/v) can be added to enhance the signal.

         Approximate recommended sample concentrations are as follow:

o        Small peptides: 1-10 pmol/L

o        Proteins: 1-50 pmol/L

o        Polymers: 1-10 mM

 

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Thermo-Fisher LCQ ESI/APCI Ion Trap

Funded by NSF grant #DBI-9729351

 

The Finnigan LCQ is a quadrupole field ion trap mass spectrometer with electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) sources. The LCQ 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.

 

LCQ Iontrap MS USER'S MANUAL in PDF format. This is the manual for a more recent LCQ. There will be some minor differences. Please read for training.


LCQ Iontrap MS Quick Guide in PDF format. This guide has been created to guide users through a standard operation of the instrument. There are screenshots and illustrations to assist. It is suggested to use this document during your analyses.


System Overview:

         Optimum mass range 50-2000 m/z, 4000 m/z in limited capacity

         Resolution of >1000 across optimum mass range on a full MS scan

         Zoom and turbo scan features

         Positive / negative ion modes

         ESI and APCI sources

         MSn (i.e. MS-MS-MS-...) mode for structural analysis

         LC-MS capability

         Protein identification with SEQUEST software

         Deconvolution tool for protein mass measurement


Calibration:

         Done by CMA staff only


Sample Preparation:

         All samples should be in an ESI-friendly solvent system such as 50/50 Water/Acetonitrile, Water/Methanol, etc.

         For positive ion samples, a small amount of acetic acid (0.1% v/v) can be added to enhance the signal.  TFA is not recommended.

         For direct injection, prepare at least 100L of sample.  For flow-injection analysis 20L is typically sufficient.

         Recommended sample concentrations are as follow:

o        Small organics: 10-100M

o        Small peptides: 5M (pmol/L)

o        Large proteins: 20-100M (pmol/L)

o        Polymers: 100-1000M

 

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Waters Q-TOF II ESI/APCI Quadrupole-TOF

 

The Micromass Q-TOF II is a hybrid mass spectrometer designed specifically for high performance in the mass range 100-2000 m/z, especially with protein identification in mind.  Samples are introduced via ESI, APCI, or Nanospray ionization sources, analyzed (optionally) by the quadrupole, fragmented (optionally) in the hexapole collision cell, then accelerated orthogonally into the TOF tube for high-resolution mass analysis.  The Q-TOF II is capable of MS and MS-MS analysis and achieves resolution exceeding 10,000.  It can also be used to detect ions above 2000 m/z although not specifically optimized to do so.

 

System Overview:

         Optimum mass range 2-2000 m/z (theoretically 2-32,000 m/z)

         Quadrupole and orthogonal V-reflectron TOF analyzers

         Positive / negative ion modes

         ESI, APCI, and nanospray ion sources

         Auto-injector valve for flow-injection analysis

         Z-spray source inlet configuration for reduced noise & contamination

         MS and MS-MS (CID) modes

         Resolution exceeding 10,000 within optimum mass range

         Femtomole-level sensitivity within optimum mass range

         5ppm mass accuracy with proper internal reference over 150-900 m/z

 

Calibration:

         Must be performed daily by individual users.

 

Sample Preparation:

         All samples should be in an ESI-friendly solvent system such as 50/50 water/acetonitrile, water/methanol, etc.

         For positive ion samples, a small amount of acetic acid (0.1% v/v) can be added to enhance the signal.  TFA is not recommended.

         For direct injection, prepare at least 100L of sample.  For flow-injection analysis 20L is typically sufficient.

         Approximate recommended sample concentrations are as follow:

o        Small organics: 1-100M

o        Small peptides: 1-20M (pmol/L)

o        Large proteins: 2-100M (pmol/L)

o        Polymers: 100-1000M

 

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