Mass Spectrometry

The CMA has five mass spectrometers available for general use:

Thermo Exactive Plus EMR ESI/nESI/DART Orbitrap Mass Spectrometer + Vanquish Flex Binary UHPLC

Thermo LTQ XL ESI/nESI/DART Linear Ion Trap + EASY-nLC 1200 nanoLC

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 Exactive Plus EMR Orbitrap Mass Spectrometer
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Thermo Scientific Vanquish Flex Binary UHPLC

The Thermo Scientific Exactive Plus EMR is a Orbitrap-based electrostatic ion trap mass spectrometer with electrospray (ESI), nanoelectrospray (nESI), and direct analysis in real time (DART) ionization capabilities. The Exactive Plus EMR has high resolution and accurate mass (HRAM) capabilities, providing high confidence molecular weight determinations. This mass spectrometer can provide molecular weight information for small molecules 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 Vanquish Flex Binary UHPLC with precise gradient flow from 0.001-8 mL/min with pressures up to 15,000 psi.

Exactive Plus EMR Product Brochure in PDF format. Please read for prior to instrument inquiries.

Click here for an Exactive Plus EMR system overview courtesy of Planet Orbitrap.

System 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)

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 or formic acid (0.1% v/v) can be added to enhance the signal.  TFA is not recommended.

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

·         Recommended sample concentrations are as follow:

o        Small organics: 1-10µM

o        Small peptides: 1-10µM (pmol/µL)

o        Large proteins: 2-10µM (pmol/µL)

o        Polymers: 10-100µM

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.

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Thermo Scientific LTQ-XL Linear Ion Trap Mass Spectrometer
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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 µL/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 400µL/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.

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 or formic acid (0.1% v/v) can be added to enhance the signal.  TFA is not recommended.

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

·         Recommended sample concentrations are as follow:

o        Small organics: 1-10µM

o        Small peptides: 1-10µM (pmol/µL)

o        Large proteins: 2-10µM (pmol/µL)

o        Polymers: 10-100µM

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 µm 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, M_n, M_w, M_z 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 1µL 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/µL

o        Proteins: 1-50 pmol/µL

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 or formic acid (0.1% v/v) can be added to enhance the signal.  TFA is not recommended.

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

·         Recommended sample concentrations are as follow:

o        Small organics: 10-100µM

o        Small peptides: 5µM (pmol/µL)

o        Large proteins: 20-100µM (pmol/µL)

o        Polymers: 100-1000µM

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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 100µL of sample.  For flow-injection analysis 20µL is typically sufficient.

·         Approximate recommended sample concentrations are as follow:

o        Small organics: 1-100µM

o        Small peptides: 1-20µM (pmol/µL)

o        Large proteins: 2-100µM (pmol/µL)

o        Polymers: 100-1000µM

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