Protein Identification, by tryptic digestion and mass spectrometry

The masses of peptides produced by the tryptic digestion of proteins are measured by mass spectrometry with high accuracy (known as a peptide mass fingerprint). Selected peptides are fragmented inside the mass spectrometer (tandem MS) to produce daughter ions, the masses of which relate to the peptide's sequence. The combined data are matched against protein sequence databases to determine the protein's identity.

Protein from 1D or 2D gels, or otherwise purified.
The protein's thiols may be blocked with iodoacetamide or acrylamide prior to SDS-PAGE.
Coomassie stains are preferable although some silver stains may be used. To minimise contamination by human keratins, wear gloves when handling samples and keep gel tanks clean and covered.

Typically, we block the protein's thiols with iodoacetamide and digest the protein in-gel with trypsin. Peptides are extracted with combinations of acid and organic solvents, concentrated by evaporation, and subjected to mass spectrometry.

Several of the APC's mass spectrometers can be used.

LC ESI MS/MS: peptides are peptides are resolved by reversed phase nanoLC into the ION-TRAP via a nanoelectrospray ion source. Peptide ions are fragmented by Collision-Induced Dissociation or Electron Transfer Dissociation.

Ion Trap: nanoLC (Agilent Technologies), CHIP Cube (Agilent Technologies, HCT Ultra ion trap (Bruker Daltonics);

Orbitrap (Thermo Fisher Scientific): nanoLC Ultimate 3000 (Dionex), LTQ Orbitrap XL ETD (Thermo Fisher Scientific): for high resolution and high mass accuracy precursor spectra in combination with fast MS/MS analysis

ION-TRAP data are processed with Bruker DataAnalysis and BioTools then searched over the in-house MASCOT-database, matching against SwissProt, MSDB or NCBI databases.

Orbitrap data are processed using Proteome Discoverer, then searched over the in-house Mascot-database or sequest database, matching against SwissProt, MSDB or NCBI databases.

MALDI TOF/TOF data are simplified to lists of monoisotopic masses and usually analysed using an in-house MASCOT server, matching against SwissProt, MSDB or NCBI databases.

A written report is provided, describing the analysis undertaken and outlining the evidence for a protein's identification. If no hit is obtained, an indication of the possible reasons may be included. The report is normally sent by email, as PDF. Raw data are in proprietary formats and are not included with the report. Data are archived by the APC.
If the data are used as part of a scientific publication, due reference to the APC should be made.

Biomolecule mass measurement, by mass spectrometry

The masses of molecules of interest (proteins/peptides, oligosaccharides, lipids or nucleic acids) can be determined with high accuracy using mass spectrometry. The size of a known protein or peptide can be confirmed or the extent of its modification (proteolytic, post-translational or chemical) determined. Mass profiles of biological fluids can be made.

The sample should be in soluble form and free of detergents. Desalting by reversed phase chromatography (on-line or off-line) is often necessary.

Several of the APC's mass spectrometers can be used.

Electrospray: generates multiple charge states of proteins

LTQ Orbitrap XL ETD (Thermo Fisher Scientific)
Ion-Trap (Bruker HCT ultra ION-Trap)
accurate to within 50-100 ppm for proteins
resolution near theoretical limit of isotopic envelopes.

MALDI: generates singly or doubly charged forms of most proteins

MALDI TOF/TOF (Bruker ultraflex III)
accurate to within 150-300 ppm for proteins
resolution ¼ - ½ of the theoretical limit of isotopic envelopes
can be highly sensitive
can generate profiles from biological fluids in a single analysis
highly accurate for the measurement of peptides of mass < 4 kDa.

Sample is introduced into electrospray instruments by direct injection or reversed phase LC.
Sample is mixed with matrix on a target plate and may be desalted on the target.

Electrospray: mass(es) are calculated by mathematical transformation of the m/z ion series.
MALDI: Masses of the analytes are normally observed directly.

A written report is provided, describing the analysis undertaken and mass(es) measured. Where appropriate, comparisons with calculated masses are made. Raw data are in proprietary formats and are not included with the report. Data are archived by the APC.
If the data are used as part of a scientific publication, due reference to the APC should be made.

Accurate mass measurement using LTQ Orbitrap MS

Compound masses can be measured with high resolution and high mass accuracy using the LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific). Accurate mass measurement allows for calculation and elucidation of the elemental composition. The high resolution is necessary to separate ions that are interfering (eg same nominal mass, but different fractional masses).
The mass spectrometer can be used with an ESI source or nanoESI source which can accommodate for flow rates from 100 nL up to several 100 uL, as well as offline nanospray.
The instrument is capable of MSn experiments (several fragmentation modes are possible, eg CID and ETD).

The sample should be of high purity. Any ionisable impurity may suppress ionization of the compound of interest and therefore poor quality spectra may result and elemental composition may not be possible to determine.
Samples can be submitted as solids or in solution using an appropriate solvent, an accurate amount and concentration of the sample must be provided. It is necessary that a low percentage of acid will be added to the sample prior to measurement. 

LTQ Orbitrap XL mass spectrometer 
accurate to within 2 ppm for most compounds
resolution better than 60 000 at m/z 400 at 1 scan/second
mass range 50-2000 or 2000-4000
Fragmentation CID, PQD or ETD

Sample introduction by ESI or nanoESI is performed by direct injection using a syringe pump. Offline nanospray is also available using coated glas needles (for up to 10 uL sample).

The mass(es) can be compared with a theoretical spectrum of the compound of interest.
The elemental composition can be calculated.

A written report is provided, describing the analysis undertaken and mass(es) measured. Comparisons with calculated mass(es) are made. Raw data are not included in the report. Data is archived by the Adelaide Proteomics Centre for at least 12 months or according to customer's suggestions.
If the data is used as part of a scientific publication, due reference to the Adelaide Proteomics Centre should be made.