The database presents experimentally verified peptides digested from the solvent-exposed domains of membrane proteins.

The peptides are created by trypsin proteolysis of membrane proteins in their host membranes, which are immobilized using the Nanoxis LPI™ FlowCell technology. Proteins are retained in their host membrane, and proteolysis occurs only outside of the lipid bilayer in contrast to in-gel digestion or in-solution digestion in the presence of detergent.

By using LPI™ technology and trypsin digestion we gather information about which parts of transmembrane proteins are outside the lipid bilayer and which of the peptides in the digest are reproducibly detectable by mass spectrometry. The peptides identified using this technology are therefore linear epitope candidates for antibody production and as reference peptides for membrane protein quantitation by mass spectrometry.

The database is useful for the following applications:

• Determining linear epitopes for antibody production
• Determining reference peptides for protein quantitation measurements by mass spectrometry
• Verifying accessibility of tryptic cleavage sites in transmembrane proteins, which can assist topology prediction

Validation of linear epitopes for antigen production

The traditional method for selecting a peptide as an immonogen candidate uses predictive algorithms to calculate physicochemical features such as hydrophobicity, secondary structure and surface exposure. A peptide selected by prediction may turn out to be a poor candidate if it is in fact membrane-anchored, poorly water soluble, or has one or more unknown post-translational modifications in vivo. Using LPI™ technology we produce peptide candidates that are solvent-exposed, easily digested, fully water soluble. Peptides identified using the LPI™ FlowCell and LC-ESI-LC-MS/MS can be used to validate immunogen candidates for antibody production.

Reference peptides for quantitative proteomics

For absolute quantification of transmembrane proteins by mass spectrometry it is important to select a suitable reference peptide from the protein of interest. Good peptide candidates are solvent-exposed, easily digested, chemically stable and readily detected by mass spectrometry. Using the LPI™ FlowCell and LC-ESI-LC-MS/MS one can produce candidate peptides that fulfil these criteria, and validate candidates chosen by prediction methods.

Verifying accessibility of tryptic cleavage sites to assist topology prediction

LPI™ technology will preserve the natural lipid environment of membrane proteins. Topology mapping is done with peptides from extramembranous domains. Peptides are generated by digestion of correctly folded membrane protein in their natural lipid environment. Topology prediction is made from experimental data together with theoretical topology prediction models, such as TMHMM.

Peptides presented in the VAEPPE database were produced and identified as follows.

Cell culture and membrane preparation

Membranes are produced from cultured cells, using standard membrane preparation methods including high-speed ultracentrifugation to wash and pellet membranes. Membranes are normally washed with Na2CO3 (pH>11) to release non-anchored proteins, thus improving the detection probability for transmembrane proteins.

Membrane protein digestion using the LPI™ FlowCell technology

Membranes are surface-immobilized using the LPI™ FlowCell technology. The immobilized membrane proteins are digested with trypsin to release peptides into solution. Following digestion the peptides are eluted from the LPI™ FlowCell.

Mass spectrometry analysis of peptides

Peptides produced using the LPI™ FlowCell are acidified and concentrated by SpeedVac.

For the liquid chromatography, an Agilent 1100 binary pump is used. The peptides are first trapped on a precolumn (45 x 0.075 mm i.d.) packed with 3 µm C18-bonded particles, followed by separation on a 200 x 0.05 mm i.d. fused silica column packed with 3 µm ReproSil-Pur C18 AQ particles. A acetonitrile gradient with 0.2 % formic acid is used for separation of the peptides and the flow through the column is approximately 100 nL/min.

Mass analyses were performed in a 7-Tesla LTQ-FT mass spectrometer (Hybrid Linear Trap Quadrupole - Fourier Transform) (Thermo Electron) equipped with a nanospray source. The instrument is operated in the data-dependent mode to automatically switch between MS and MS/MS acquisition. MS spectra are acquired in the FT-ICR while MS/MS spectra are acquired in the LTQ-trap. For each MS scan, the six most intense, doubly or triply charged ions are sequentially fragmented in the linear trap by collision induced dissociation (CID). Fragmented target ions are excluded for MS/MS selection for 6 seconds.

All tandem mass spectra are searched by MASCOT (Matrix Science) against Sprot or NCBInr. Standard search settings are: enzyme = trypsin; variable modifications = oxidation (M); peptide mass tolerance = 5 ppm; fragment mass tolerance = 0.5 Da; max missed cleavages = 1.

The database presents only peptides that fulfill criteria used for reference peptides for quantitation by mass spectrometry.

The peptides fulfill the following criteria:

1) Length = 8-20 amino acids

2) Contain no reactive residues (C,M,W)

3) Contain no unstable residues (NG, DG,QG,N-term N, N-term Q)

4) Contain no C-terminal KK, RR, KR or RK (ragged-ends)

5) Contain no missed cleavage sites

6) Are peptides from transmembrane proteins