Experimental, Proteomics
Nano LC: Analyts are separated using C18 reversed phase column at 250-300nL/min by increasing the concentration of Buffer B (80% Acetonitrile [OPTIMA LC/MS, A955, Fisher Scientific]/20% water [OPTIMA LC/MS, W61, Fisher Scientific] /0.1% formic acid [OPTIMA LC/MS, A11750, Fisher Scientific]) while decreasing the concentration of Buffer A (1% Acetonitrile [OPTIMA LC/MS, A955, Fisher Scientific]/99% water [OPTIMA LC/MS, W61, Fisher Scientific] /0.1% formic acid [OPTIMA LC/MS, A11750, Fisher Scientific]) over a time period.
Pre-column setup: Analyts are loaded on to a pre-column (100um x 2cm, 164564, THERMO FISHER SCIENTIFIC) allowing for concentration and desalting. The pre-column is hereafter connected to an analytical column and the analyts are eluted and separated
.
Direct loading setup: Analyts are concentrated and desalted off-line and sample is loaded directly onto the analytical column using 100% Buffer A.
Analytical packed-in-emitter C18 column, 100um inner diameter: The analytical column (15cm) is packed in a pulled emitter (NTCC-360/100-5-153, NIKKYO TECHNOS CO., LTD.)
Analytical packed-in-emitter C18 column, 75um inner diameter: The analytical column (12cm) is packed in a pulled emitter (NTCC-360/75-5-123, NIKKYO TECHNOS CO., LTD.)
Analytical heated C18 column: length of 25cm and inner diameter of 75um (ES902, Easy Spray Pepmap RSLC C18um, THERMO ELECTRON NORTH AMERICA LLC.)
Digestion in 8M urea:
Micro-SPE: Empore C18 (3M) are activated (80% Acetonitrile [OPTIMA LC/MS, A955, Fisher Scientific]/20% water [OPTIMA LC/MS, W61, Fisher Scientific] /0.1% trifluroacetic acid [LCMS Grade, 85183, THERMO FISHER SCIENTIFIC). Membranes are conditioned (1% Acetonitrile [OPTIMA LC/MS, A955, Fisher Scientific]/99% water [OPTIMA LC/MS, W61, Fisher Scientific] /0.1% trifluroacetic acid [LCMS Grade, 85183, THERMO FISHER SCIENTIFIC) and samples are loaded in aqueous solvent. Membranes are washed in condition buffer and analyts are eluted using (60% Acetonitrile [OPTIMA LC/MS, A955, Fisher Scientific]/20% water [OPTIMA LC/MS, W61, Fisher Scientific] /0.1% trifluroacetic acid [LCMS Grade, 85183, THERMO FISHER SCIENTIFIC).
Large SPE
Solvents
Mass spec
Type of digestion
Search engine
Quantitation
Data base: Data bases of choice are by UniProt. Main data bases are concatenated with relevant protein contaminants. For samples originating from tissue, serum, plasma, blood the main data base is concatenated with the sequences of the enzymes used to digest the samples. For samples originating from cell culture where Fetal Bovine Serum is used, the main data base concatenated with common bovine contaminating proteins. For very targeted analysis or specific protein sequence we can decided to search the data against the target protein concatenated with a smaller proteome.
Acetone precipitation: Ice cold acetone () precipitation to remove non-ionic detergent.
Wessel & Fluegge precipitation: To an aqueous sample is added methanol (OPTIMA LC/MS, W456, Fisher Scientific]) chloroform (HPLD plus, 650471, Sigma) and water (OPTIMA LC/MS, W61, Fisher Scientific). After vortex the mixture is spun where after the top layer is removed and additional methanol is added. Proteins are precipitated by centrifugation.
PRM