QUALITY

Reliability Code TRANSPATH presents more than an extraction of data from the published literature. It also includes a quality evaluation and a reliability code. The reliability of experimental evidence in the sense of biological relevance is defined by the source of the biological material and the method used for indication (    Table 1). Our annotators evaluate the quality of experimental data from primary literature. The reliability code given in the quality matrix (    Table 2) is a combination from a list of methods and a category of the biological material. Biological material can be extracted from a complete organism, for example a knock-out mutant, or recombinant polypeptides. Thus, reactions obtained from experiments with different biological materials have different probabilities for existing in vivo. We provide a reliability value that is defined by each possible combination of category and method on a scale of 1 to 5 (highest to lowest reliability,    Table 3). The value is attached to mechanistic reactions. The annotators use only data from peer-reviewed publications. If controls are missing or are not credible in a specific paper, the data will not be included.

Table 1: Methods and biological material: abbreviations and classification

	Material		Method
A1	Wild-type	M1	Yeast two-hybrid system
A2	Natural chromosomal mutants leading to new phenotype	M2	Screening of expression libraries with other (labeled) proteins (e.g. phage display, eukaryotic cDNA library with EXIox vectors)
A3	Knock-out	M3	(Co-) localization of proteins in cells and tissues by microscopical analysis (e.g. immunofluorescence, in situ detection, electron microscopy)
A4	Transgenic animals	M4	Affinity precipitation in solution/batch (e.g. with sepharose or magneto beads) e.g. immunoprecipitation, pull-down assay
A5	Induced, but not defined mutations	M5	In vitro protein-protein binding assay (solid phase) e.g. ELISA (Enzyme Linked Immuno Sorbent Assay)
B1	Embryonic cells	M6	BIACORE (detection and quantification of ligands)
B2	Stem cells -> pluripotent	M7	Far Western or Gel overlay (detection of binding partners on blotted proteins after transfer from a gel) or protein-lipid overlay assay
B3	Primary cells	M8	Affinity chromatography with matrices (proteins coupled to e.g. sepharose or agarose matrices)
B4	Recombinant cells/tissue/organisms (transfected); not permanent cell lines	M9	Peptide spot (search for binding partners of defined peptide motifs on a membrane)
C1	Permanent tissue culture cells	M10	Crosslinking (detection by SDS-Page and Western blot)
D1	Permanent or transient transfected or infected cells (tissue culture cells)	M11	EMSA (electrophoretic mobility shift assay)
D2	Recombinant proteins produced e.g. from Baculo virus vectors in insect cells, transfected/microinjected Xenopus oocytes	M12	Binding assay (binding of labeled ligand to receptor-expressing cells, quantitative evaluation, for example Scatchard analysis)
E1	Recombinant proteins from Yeast	M13	Modification of proteins in living cells (e.g. proteinphosphorylation and -dephosphorylation, ubiquitination, farnesylation; detection of the modified proteins, e.g. from cell extracts)
F1	Recombinant proteins from e.g. E.coli, Bacillus subtilis	M14	Subcellular extraction of proteins (fractionation for localization of proteins)
F2	In vitro translation, synthetic peptides	M15	Dot spots (spotted proteins detected with specific antibodies)
		M16	In vitro modification of a substrate (e.g. phosphorylation or dephosphorylation (e.g. Kinase-assay), ubiquitination)
		M17	Reporter gene assay (luciferase assay, beta-galactosidase)
		M18	Western blot, SDS page
		M19	mRNA detection by Northern blot (indirect evidence for protein interaction)
		M20	Localization of mRNA in tissues by e.g. FISH, ISH (p32, H3) (indirect evidence for protein interaction)
		M21	Detection of mRNA by RT-PCR (reverse transcriptase, thereafter PCR; quantitative mRNA detection) (indirect evidence for protein interaction)
		M22	Mammalian two (or tri) -hybrid assay
		M23	Co-sedimentation and -purification by chemico-physical methods (biochemical purification of stable complexes by e.g analytical ultracentrifugation, size-exclusion chromatography (SEC))
		M24	Protein cleavage and specific degradation (documented by protein detection of e.g. S35-labeled or biotinylated protein fragments, immunoblots, SDS-PAGE only, HPLC, mass spectral analysis)
		M25	Interaction measurement by chemico-physical methods (e.g. crystal structure analysis of complexes; spectrofluorometer FRET: fluorescence resonance energy transfer; intensity and/or wavelength of fluorescence of a given interacting compound changes, when another molecule binds to it; quantitative; usually done in vitro; also NMR: looking at changes in nuclear spin, usually done with peptide fragments and in unphysiological solutions, isothermal titration calorimetry (ITC))
		M26	mRNA expression profile analysis (e.g. microarray data)

Table 2: Quality matrix

	A1-5	B1-3	B4	C1	D1	D2	E1	F1-2
M1	0	0	0	0	0	0	2	0
M2	0	0	0	0	0	0	5	5
M3	2	3	3	3	3	4	4	0
M4	1	2	2	2	2	3	4	5
M5	0	2	3	3	4	4	4	5
M6	3	3	3	3	3	3	3	4
M7	3	3	3	3	4	4	4	5
M8	3	3	4	4	4	5	5	5
M9	4	4	4	4	4	4	4	4
M10	2	2	3	3	3	4	5	5
M11	4	4	4	4	4	4	4	4
M12	3	3	3	3	3	4	4	4
M13	2	2	2	2	3	4	4	5
M14	3	3	3	3	3	0	0	0
M15	3	3	3	3	3	3	4	5
M16	3	3	3	3	3	4	4	5
M17	0	0	3	0	3	3	3	4
M18	2	2	2	2	2	3	3	3
M19	3	3	4	4	4	4	4	5
M20	2	0	0	0	0	0	0	0
M21	4	4	4	4	4	4	4	0
M22	0	0	0	0	1	0	0	0
M23	3	3	3	3	3	4	4	5
M24	3	3	3	3	3	4	4	5
M25	2	2	2	2	3	3	4	5
M26	4	4	4	4	5	0	0	0

Table 3: Quality scale: reliability value

priorities	reliability
1	highest reliability
2	high reliability
3	moderate (average) reliability
4	modest reliability
5	low reliability
0	value not assigned