TRANSPATH Professional Documentation

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 Description/Examples

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 Surface plasmon resonance Detection and quantification of ligands

B2 Stem cells -> pluripotent M7 Far Western or Gel overlay or protein-lipid overlay assay Gel overlay: detection of binding partners on blotted proteins after transfer from a gel

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 E.g. 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

M27 Transmembrane potential measurements E.g. current-voltage experiments
M28 Predicted by binding-site sequence
M29 Co-localization
M30 In vitro binding
M31 In vitro reconstitution of activity
M32 Flow cytometry
M33 Ligand binding
M34 Two-hybrid Cell system not specified

	Material		Method	Description/Examples
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	Surface plasmon resonance	Detection and quantification of ligands
B2	Stem cells -> pluripotent	M7	Far Western or Gel overlay or protein-lipid overlay assay	Gel overlay: detection of binding partners on blotted proteins after transfer from a gel
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	E.g. 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
		M27	Transmembrane potential measurements	E.g. current-voltage experiments
		M28	Predicted by binding-site sequence
		M29	Co-localization
		M30	In vitro binding
		M31	In vitro reconstitution of activity
		M32	Flow cytometry
		M33	Ligand binding
		M34	Two-hybrid	Cell system not specified

Table 2: Quality matrix

A1-5 B1-3 B4 C1 D1 D2 E1 F1-2

M1 6 6 6 6 6 6 2 6

M2 6 6 6 6 6 5 5 5

M3 2 3 3 3 3 4 4 6

M4 1 2 2 2 2 3 4 5

M5 2 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 4 5 5

M15 3 3 3 3 3 3 4 5

M16 3 3 3 3 3 4 4 5

M17 6 3 3 3 3 3 3 4

M18 2 2 2 2 2 3 3 3

M19 3 3 4 4 4 4 4 5

M20 2 6 6 6 6 6 6 6

M21 4 4 4 4 4 4 4 6

M22 6 6 6 6 1 6 6 6

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 6 6 6

M27 3 3 3 4 4 4 4 4

M28* 6 6 6 6 6 6 6 6

M29* 6 6 6 6 6 6 6 6

M30* 6 6 6 6 6 6 6 6

M31* 6 6 6 6 6 6 6 6

M32* 6 6 6 6 6 6 6 6

M33* 6 6 6 6 6 6 6 6

M34* 6 6 6 6 6 6 6 6

*Methods were added in the course of integration of reactions from Proteome BKL. Quality values have not yet been assigned, because the integrated reactions lack the material information yet.

Table 3: Quality scale: reliability value

priorities reliability

1 highest reliability

2 high reliability

3 moderate (average) reliability

4 modest reliability

5 low reliability

6 value not assigned

	A1-5	B1-3	B4	C1	D1	D2	E1	F1-2
M1	6	6	6	6	6	6	2	6
M2	6	6	6	6	6	5	5	5
M3	2	3	3	3	3	4	4	6
M4	1	2	2	2	2	3	4	5
M5	2	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	4	5	5
M15	3	3	3	3	3	3	4	5
M16	3	3	3	3	3	4	4	5
M17	6	3	3	3	3	3	3	4
M18	2	2	2	2	2	3	3	3
M19	3	3	4	4	4	4	4	5
M20	2	6	6	6	6	6	6	6
M21	4	4	4	4	4	4	4	6
M22	6	6	6	6	1	6	6	6
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	6	6	6
M27	3	3	3	4	4	4	4	4
M28*	6	6	6	6	6	6	6	6
M29*	6	6	6	6	6	6	6	6
M30*	6	6	6	6	6	6	6	6
M31*	6	6	6	6	6	6	6	6
M32*	6	6	6	6	6	6	6	6
M33*	6	6	6	6	6	6	6	6
M34*	6	6	6	6	6	6	6	6

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