Protein Chemistry Section
Mass Spectromety: Technology
The Mass Spectrometry laboratory is equipped with four mass spectrometers that have both complimentary and overlapping capabilities for the characterization and identification of peptides, proteins, and other biological compounds. The types of analyses and technical support presently available and forthcoming are briefly described below.
Analyses and Technical Support
Molecular Mass Determinations of Organic Molecules, Peptides, Protein Digests, and Intact Proteins (API 365 TGMS)
- Electrospray Ionization and Direct Infusion
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- Small ionizable molecules
- Semi-purified and pure bio-molecules
- Limited peptide and protein mixtures
- Chromatography-Coupled Electrospray
- Samples with salt and other contaminants
- Complex mixtures and digests not amenable to direct infusion
- Automated High Throughput Mass Analyses by Electrospray
- Samples without detrimental salts by direct flow injection
- Samples with salts removable with reverse phase trapping
- MALDI
- Crude mixtures not amenable to electrospray
- Tryptic digest analysis
Other Techniques (Presently Require Picomolar Amounts)
- Estimation of peptide and protein purity by electrospray
- Disulfide bond analysis
- Chemically modified amino acids
- MS/MS product ion analysis for structural information
Q-TOF2 Hybrid MS for Protein Identification and Characterization (Shared Instrumentation, Bldg. 8)
- Identification of proteins by peptide map LC/MS and combined LC/MS/MS (amino acid sequence data) from complex sources
- Presently analyses are available for gel extracted protein digests and solution digests at low picomole amounts
- Amenable to extensive peptide mapping of protein digests
- Suited for de novo peptide sequencing
QSTAR XL
- Flexible MS/MS platform for both nano-electrospray coupled with LC Packings nano-flow HPLC system and o-MALDI analysis for rapid femtomole sensitivity protein identification
- Suited for de novo peptide sequencing and extensive peptide mapping of protein digests
- High sensitivity and accurate mass accuracy yield unequivocal molecular weights and protein structural information
- Bioanalyst, Pro ID, and Pro ICAT software for advanced and automated analysis of mass spectrometry data
Instruments
Electrospray (ES) Mass Spectrometer (MS) System (API 365 TQMS)
The electrospray (ES) mass spectrometer (MS) system, Applied Biosystems (AB)-Sciex triple quadrupole mass spectrometer (API 365 TQMS) is supported with a Perkin Elmer (PE) 140C Microgradient HPLC system and a PE Series 200 Autosampler. It is ideal for small molecule, peptide, and protein mass analysis, characterization, and quantification.
Resolution ~ 1000
Mass Accuracy ~ 100 ppm
Sensitivity ~ 0.1-1 picomole
AB Voyager RP Matrix-assisted Laser Desorption Ionization (MALDI)—Time of Flight MS
The Voyager MALDI TOF MS is a more specialized standalone instrument suitable for peptide QC and non-fractionated protein digests.
Resolution ~ 2500 @ 1000 amu
Mass Accuracy ~ 100 ppm (internal standard)
Sensitivity ~ 0.05-1 picomole
Micromass Electrospray Quadropole—Orthogonal Time of Flight (Q-Tof2) MS
The Q-Tof2 MS is part of a joint, five-Institute proteomics facility. Computer controlled data dependent automated switching to MS/MS provides peptide sequence information. Suited for online liquid chromatography-mass spectrometry (LC/MS and LC/MS/MS) for peptide sequencing (also de novo sequencing), protein identification, and characterization. This MS has an extensive software package for proteomic analyses.
Resolution ~ 9,000 @ 1000 amu
Mass Accuracy ~ 5ppm
Sensitivity ~ low femtomoles
AB Qstar XL Hybrid Q-TOF LC/MS/MS System
The QSTAR XL is an in-house mass spectrometer located in the Twinbrook I facility. The QSTAR XL is ideal for online nano-flow liquid chromatography (with an LC Packings Ultimate nano-flow HPLC system)—mass spectrometry (LC/MS and LC/MS/MS) for demanding peptide sequencing (also de novo sequencing), protein identification, and characterization. Allows switching to o-MALDI for sensitive, high-throughput, more routine work. Extensive software package is available for automated and advanced analysis of mass spectrometry data.
Resolution ~ 9,000 @ 1000 amu
Mass Accuracy ~ 5ppm
Sensitivity ~ low femtomoles
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