Table 2. Proteins interacting with DmHsp22 by orbitrap fusion LC-MS/MS mass spectrometer.
| Detected proteins | IAC on S1300 of HeLa cells | |||||
| Detected proteins (Accession number) | First IAC | Second IAC | ||||
| DmHsp22-Flag | Control | DmHsp22-Flag | Control | Heat Shock +6 h post-recovery | ||
| ADP/ATP translocase2, SLC25A5 (P05141) | 27.5 | 3.33 | 9.13 | 1 | 4 | |
| ADP/ATP translocase3, SLC25A6 (P12236) | 16.2 | 1.5 | 7.26 | 0.78 | 3.18 | |
| Isoform 2 of ATPase family AAA domain-containing protein 3A (Q9NV17-2) | 12.4 | 0.6 | 4.81 | 1 | 2 | |
| ATPase family AAA domain-containing protein 3B (Q5T9A4) | 5.11 | 0 | 0 | 0 | 0 | |
| ATP synthase subunit alpha, mitochondrial (P25705) | 7.2 | 2.2 | 21.5 | 1.5 | 12 | |
| ATP synthase subunit beta, mitochondrial (P06576) | 10.6 | 3.5 | 10 | 2.99 | 6.53 | |
| ATP synthase subunit g, mitochondrial (E9PN17) | 10.4 | 0.2 | 7 | 1 | 6 | |
| ATP synthase subunit gamma (P36542) | 5.5 | 0.5 | 2.5 | 0.14 | 1.3 | |
| Isoform Heart of ATP synthase subunit gamma, mitochondrial (P36542) | 4.82 | 0.5 | 2.5 | 0.1 | 2 | |
| ATP synthase subunit e, mitochondrial (P56385) | 4.51 | 0 | 8.35 | 3.6 | 6 | |
| ATP synthase subunit d, mitochondrial (O75947) | 3.86 | 0.8 | 3.5 | 1.1 | 1.85 | |
| ATP synthase subunit epsilon (P56381) | 3.65 | 0.4 | 0 | 0 | 0 | |
| ATP synthase subunit O, mitochondrial (P48047) | 2.12 | 0.8 | 3.67 | 0.64 | 3.16 | |
| ATP synthase F(0) complex subunit B1, mitochondrial (P24539) | 0.54 | 0 | 0 | 0 | 0 | |
| ATP synthase subunit delta, mitochondrial (P30049) | 0.43 | 0 | 3.47 | 1.1 | 1.99 | |
| Phosphate carrier protein, mitochondrial (F8VVM2) | 7.6 | 0.6 | 4.48 | 0.32 | 1.8 | |
| Isoform 2 of Calcium-binding mitochondrial carrier protein Aralar1, SLC25A12 (F8W9J0) | 2.2 | 0.12 | 0.91 | 0.12 | 0.84 | |
| Calcium-binding mitochondrial carrier protein SCaMC-1, SLC25A24 (Q6NUK1) | 0.13 | 0 | 0.44 | 0 | 0.23 | |
| Calcium-binding mitochondrial carrier protein Aralar2, SLC25A13 (Q9UJS0) | 3.3 | 0.15 | 1.7 | 0 | 1.12 | |
| Tricarboxylate transport protein, mitochondrial SLC25A1 (P53007) | 1 | 0.2 | 0 | 0 | 0 | |
| Mitochondrial 2-oxoglutarate/malate carrier protein, SLC25A11 (Q02978) | 1 | 0 | 1 | 0 | 1.11 | |
| Mitochondrial thiamine pyrophosphate carrier (Q9HC21) | 0.43 | 0 | 0 | 0 | 0 | |
| Mitochondrial glutamate carrier 1, SCL25A22 (Q9D6M3) | 0.35 | 0 | 0.72 | 0.14 | 0.86 | |
| Solute carrier family 25 member 46 (Q96AG3) | 0.16 | 0 | 1.8 | 0 | 0.1 | |
| 60 kDa heat shock protein, mitochondrial (P10809) | 9 | 1 | 27.8 | 6 | 16.8 | |
| Stress-70 protein, mitochondrial (P38646) | 2.65 | 0.7 | 1.32 | 0.8 | 1.23 | |
| HSP 90-beta (P08238) | 2.58 | 1.25 | 2.87 | 0.9 | 0 | |
| Elongation factor Tu, mitochondrial (P49411) | 4 | 1.27 | 19 | 7 | 16.7 | |
| 78 kDa glucose-regulated protein (P11021) | 8.74 | 1.5 | 5.5 | 2.8 | 0 | |
| Prohibitin (P35232) | 4.18 | 1 | 1.4 | 1.7 | 0 | |
| Probibitin-2 (J3KPX7) | 3 | 0.6 | 1 | 1 | 1.52 | |
| Glutamate dehydrogenase 1, mitochondrial, (P00367) | 2.4 | 0 | 2.85 | 0.1 | 0.76 | |
| Voltage-dependent anion-selective channel protein 1 (P21796) | 4 | 0 | 1.32 | 0.39 | 2.69 | |
| Voltage-dependent anion-selective channel protein 2 (A0A0A0MR02) | 1.15 | 0.23 | 0 | 0 | 0 | |
| Mitochondrial import receptor subunit TOM22 (Q9NS69) | 1.8 | 0.51 | 0 | 0 | 0 | |
| Mitochondrial import receptor subunit TOM70 (O94826) | 0.1 | 0 | 0 | 0 | 0 | |
| Mitochondrial import inner membrane translocase subunit TIM50 (Q3ZCQ8) | 0.84 | 0 | 1.78 | 0 | 1 | |
| Mitochondrial import inner membrane translocase subunit TIM14 (Q96DA6) | 0.6 | 0 | 0 | 0 | 0 | |
| Mitochondrial import inner membrane translocase subunit TIM44 (O43615) | 0.45 | 0 | 0 | 0 | 0 | |
| Mitochondrial import inner membrane translocase subunit TIM23 (O14925) | 0.34 | 0 | 3 | 0 | 3 | |
| Detected proteins | IAC on S1300 of HeLa cells | |||||
| Detected proteins (Accession number) | First IAC | Second IAC | ||||
| DmHsp22-Flag | Control | DmHsp22-Flag | Control | Heat Shock +6 h recovery | ||
| Putative mitochondrial import inner membrane translocase subunit Tim23B (Q5SRD1) | 0 | 0 | 1.18 | 0 | 0.44 | |
| MICOS complex subunit MIC60 (B9A067) | 1.55 | 0 | 0.78 | 0 | 0.25 | |
| MICOS complex subunit MIC13 (Q8R404) | 1.31 | 0.1 | 4.73 | 0 | 2.57 | |
| MICOS complex subunit MIC27 (Q6UXV4) | 0.25 | 0 | 0 | 0.14 | 0 | |
| MICOS complex subunit MIC19 (Q9NX63) | 1.34 | 0.38 | 4.16 | 0 | 1.44 | |
| MICOS complex subunit MIC 25 (Q9BRQ6) | 0 | 0 | 4.16 | 0 | ||
| Cytochrome b-c1 complex subunit 2, mitochondrial (P22695) | 1 | 0 | 1.46 | 0.4 | 0.9 | |
| Cytochrome b-c1 complex subunit Rieske-like protein 1 (P47985) | 0.24 | 0 | 0 | 0 | ||
| Cytochrome c oxidase subunit 6C (P09669) | 0.88 | 0 | 1.39 | 0 | 0 | |
| Cytochrome c oxidase assembly factor 3 homolog, mitochondrial (Q9Y2R0) | 1.5 | 0 | 0 | 0 | ||
| Cytochrome c oxidase subunit NDUFA4 (O00483) | 0.83 | 0 | 0 | 0 | 0 | |
| Cytochrome c oxidase subunit 2 (P00403) | 0.62 | 0 | 0 | 0 | 0 | |
| Cytochrome c1, heme protein, mitochondrial (P08574) | 0.43 | 0.19 | 0.67 | 0 | 0.48 | |
| Cytochrome c oxidase subunit 4 isoform 1, mitochondrial (P13073) | 0.38 | 0 | 1.4 | 0 | 0 | |
| Ubiquinol-cytochrome-c reductase complex assembly factor 3 (Q6UW78) | 0.76 | 0 | 0 | 0 | ||
| NADH-cytochrome b5 reductase 3 (P00387) | 0.45 | 0 | 0 | 0 | ||
| NADPH-cytochrome P450 reductase (E7EMD0) | 0.15 | 0.04 | 0.15 | 0 | ||
| NADH dehydrogenase 1 alpha subcomplex subunit 9, mitochondrial (Q16795) | 1 | 0.16 | 0 | 0 | ||
| NADH dehydrogenase 1 beta sub-complex subunit 10 (H3BPJ9) | 0.84 | 0.11 | 0 | 0 | 0 | |
| NADH dehydrogenase 1 beta sub-complex subunit 6, mitochondrial (O95139) | 0.5 | 0.14 | 0 | 0 | ||
| NADH dehydrogenase iron-sulfur protein 8, mitochondrial (E9PN51) | 0.61 | 0 | 0 | 0 | 0 | |
| NADH dehydrogenase iron-sulfur protein 3, mitochondrial (O75489) | 0.51 | 0 | 0 | 0 | ||
| NADH dehydrogenase 1 beta subcomplex subunit 5, mitochondrial (E7EWP0) | 0.37 | 0.1 | 0 | 0 | ||
| Succinate dehydrogenase flavoprotein subunit, mitochondrial (D6RFM5) | 0.36 | 0.14 | 0.78 | 0.23 | 0.25 | |
| NADH dehydrogenase iron-sulfur protein 7, mitochondrial (A0A087WT13) | 0.31 | 0.09 | 0 | 0 | ||
| NADH dehydrogenase 1 alpha subcomplex subunit 5 (A0A087X1G1) | 2.51 | 0 | 0 | 0 | ||
| NADH-ubiquinone oxidoreductase 75 kDa, mitochondrial (P28331) | 0.1 | 0 | 0 | 0 | ||
| NAD(P) transhydrogenase, mitochondrial (Q13423) | 0.26 | 0 | 0.19 | 0 | 0.09 | |
| NADH dehydrogenase iron-sulfur protein 2, mitochondrial (O75306) | 0.13 | 0 | 0 | 0 | ||
| Acetyl-CoA acetyltransferase (P24752) | 0.33 | 0.1 | 0 | 0 | ||
| Up-regulated during skeletal muscle growth protein 5 (Q96IX5) | 6.14 | 0.35 | 0 | 0 | ||
| Carbamoyl-phosphate synthase (ammonia), mitochondrial (P31327) | 0.7 | 0.6 | 16 | 2 | ||
| KN motif and ankyrin repeat domain-containing protein 2 (Q63ZY3) | 3 | 0 | 1.6 | 0 | ||
| Sideroflexin (A0A0A0MS41) | 1.93 | 0 | 4.4 | 0.3 | 2.78 | |
| Sideroflexin-1 (Q9H9B4) | 0.68 | 0.12 | 2 | 0.2 | 1 | |
| Trifunctional enzyme subunit alpha, mitochondrial (P409339) | 0.47 | 0.16 | 0.06 | 0 | 0.04 | |
| Trifunctional enzyme subunit beta (B5MD38) | 0.88 | 0.25 | 0 | 0 | 0 | |
| Stomatin-like protein 2, mitochondrial (Q9UJZ1) | 1.13 | 0.11 | 0.51 | 0.4 | ||
| Sulfide:quinone oxidoreductase, mitochondrial (Q9Y6N5) | 1 | 0 | 0.88 | 0 | ||
| Detected proteins | IAC on S1300 of HeLa cells | |||||
| Detected proteins (Accession number) | First IAC | Second IAC | ||||
| DmHsp22-Flag | Control | DmHsp22-Flag | Control | Heat Shock +6 h recovery | ||
| Aspartate-tRNA ligase, mitochondrial (Q6PI48) | 1 | 0 | 0.92 | 0 | ||
| Serine/threonine-protein phosphatase PGAM5, mitochondrial (Q96HS1) | 1 | 0 | 0 | 0 | ||
| Isoform 2 of Carnitine O-palmitoyltransferase1, liver isoform (P50416) | 0.86 | 0 | 0.25 | 0 | 0.08 | |
| Isoform 3 of pyruvate dehydrogenase E1 component subunit alpha, mitochondrial (P08559) | 0.54 | 0 | 0.9 | 0.33 | 0.67 | |
| Glutaryl-CoA dehydrogenase, mitochondrial (Q929470) | 0.67 | 0 | 0 | 0 | ||
| Pyruvate dehydrogenase E1 component subunit beta, mitochondrial (P11177) | 0.61 | 0.17 | 1 | 0.2 | 0.1 | |
| DNA-3-methyladenine glycosylase (P29372) | 0.47 | 0.06 | 0 | 0 | ||
| Phosphatidyl glycerophosphatase and protein-tyrosine phosphatase1 (Q8WUK0) | 0.33 | 0.1 | 0 | 0 | ||
| Methylcrotonyl-CoA carboxylase subunit alpha, mitochondrial (Q96RQ3) | 0 | 0 | 0.34 | 0 | ||
| Isoform 3 of Carbamoyl-phosphate synthase, mitochondrial (P31327) | 0 | 0 | 0.74 | 0.04 | ||
| Pyruvate carboxylase, mitochondrial (P11498) | 0 | 0.04 | 1 | 0.1 | 0.06 | |
| Hydroxysteroid dehydrogenase-like protein 2 (Q6YN16) | 0.16 | 0 | 0 | 0 | ||
| Succinyl-CoA ligase subunit beta, mitochondrial (Q96199) | 0.16 | 0 | 0 | 0 | ||
| Saccharopine dehydrogenase-like oxidoreductase (Q8NBX0) | 0.15 | 0 | 0 | 0 | ||
| Probable arginine-tRNA ligase (Q5T160) | 0.11 | 0 | 0 | 0 | ||
| Lysine-tRNA ligase (Q15046) | 0.1 | 0 | 0 | 0 | ||
| Valine-tRAN ligase (P26640) | 0.1 | 0.06 | 0.11 | 0.1 | 0.1 | |
| DnaJ homolog subfamily A member 1 (P31689) | 2 | 0.34 | 0 | 0 | ||
| Erythrocyte band 7 integral membrane protein (P27105) | 1.43 | 0.31 | 2.27 | 0.5 | ||
| Arginine and glutamate-rich protein 1 (Q9NWB6) | 1.36 | 0.4 | 0.72 | 0.76 | ||
| ATP-binding cassette sub-family F member 2, mitochondrial (Q9UG63) | 0.32 | 0.02 | 1.95 | 0.33 | 0.9 | |
| ATP-dependent Clp protease ATP-binding subunit clip-like, mitochondrial (O76031) | 1 | 0 | 0 | 0 | ||
| Putative coiled-coil-helix-coiled-coil domain-containing protein (Q9BUK0) | 0.5 | 0 | 5 | 2 | 4 | |
| Evolutionary conserved-signalin intermediate in Toll pathway, mitochondrial (K7EJG5) | 1 | 0 | 0 | 0 | ||
| Protein MTO1 homolog, mitochondrial (Q9Y2Z2) | 0.91 | 0 | 0 | 0 | ||
| Protein NipSnap homolog 1 (Q9BPW8) | 0.73 | 0 | 0.32 | 0.1 | 0.37 | |
| 60S ribosomal protein L35a (P18077) | 0.6 | 0.6 | 7 | 5.2 | ||
| Isoform 2 of HCLS1-associated protein X-1 (O00165) | 0.5 | 0 | 0.53 | 0.38 | 0.38 | |
| Isoform 4 of Clusterin (P10909-4) | 0.5 | 0 | 0 | 0 | ||
| AarF domain-containing protein kinase 4 (M0QZZ2) | 0.47 | 0.13 | 0 | 0 | ||
| V-type ATPase catalytic subunit A (P38606) | 0.45 | 0.06 | 0 | 0 | ||
| 39S ribosomal L13, mitochondrial (Q9BYD1) | 0.44 | 0 | 0 | 0 | ||
| Mitochondrial inner membrane protein OXA1L (Q15070) | 0.42 | 0 | 0 | 0 | ||
| GTPase Era, mitochondrial (O75616) | 0.31 | 0.04 | 0 | 0 | ||
| Annexin A1 (P04083) | 0.4 | 0 | 0 | 0 | ||
| Peptidyl-Trna hydrolase 2, mitochondrial (Q9Y3E5) | 0.38 | 0.11 | 0 | 0 | ||
| Complement component 1 Q subcomponent-binding protein, mitochondrial (Q07021) | 0.37 | 0 | 0 | 0 | ||
| Apoptosis-inducing factor 1, mitochondrial (O95831) | 0.34 | 0 | 1.2 | 0.1 | 0.44 | |
| Aldehyde dehydrogenase X, mitochondrial (P30837) | 0.263 | 0.07 | 0.28 | 0 | 0.13 | |
| Detected proteins | IAC on S1300 of HeLa cells | |||||
| Detected proteins (Accession number) | First IAC | Second IAC | ||||
| DmHsp22-Flag | Control | DmHsp22-Flag | Control | Heat Shock +6 h recovery | ||
| Dnaj homolog subfamily A member 3, mitochondrial (Q96EY1) (Q96EY1) |
0.3 | 0 | 0 | 0 | 0 | |
| Danj homolog subfamily C member 11 (Q9NVH1) | 0.237 | 0 | 0 | 0 | 0 | |
| Alkyldihydroxyacetonephosphate synthase (O00116) | 0.2 | 0 | 0 | 0 | 0 | |
| Succinyl-CoA ligase subunit alpha, mitochondrial (P53597) | 0.2 | 0.12 | 0 | 0 | 0 | |
| Protein-glutamine gamma-glutamyltransferase 2 (P21980) | 0.2 | 0.02 | 0.2 | 0.3 | ||
| Putative ribosome-binding factor A, mitochondrial (Q8N0V3) | 0.2 | 0.05 | 0 | 0 | ||
| NADH dehydrogenase flavoprotein 1, mitochondrial (B4DE93) | 0.18 | 0 | 0 | 0 | ||
| Dimethyladenosine transferase 1, mitochondrial (Q8WVM0) | 0.2 | 0.05 | 0 | 0 | ||
| Long-chain-fatty-acid-CoA ligase 3 (O95573) | 0.185 | 0.02 | 1.18 | 0.1 | 0.19 | |
| Synaptic vesicle membrane protein VAT-1 homolog (Q99536) | 0.17 | 0 | 0 | 0 | ||
| Glutamine-tRNA ligase (P41250) | 0.08 | 0.02 | 0 | 0 | ||
| ATP-binding cassette sub-family B member 6, mitochondrial (Q9NP58) | 0.07 | 0 | 0 | 0 | ||
| 2-oxoglutarate dehydrogenase, mitochondrial (A0A0D9SFS3) | 0.06 | 0 | 0 | 0 | ||
| Activating molecule in BECN1-regulated autophagy protein 1 (Q9C0C7) | 0.05 | 0.01 | 0 | 0 | ||
| Putative ATP-dependent RNA helicase DHX30 (H7BXY3) | 0.05 | 0.03 | 2 | 3 | ||
| Microtubule-associated tumor suppressor 1 (Q9ULD2) | 0.05 | 0 | 0.28 | 0 | ||
| Serine/threonine-protein kinase mTOR, mitochondrial (P42345) | 0 | 0 | 0.07 | 0 | ||
| Single-stranded DNA-binding protein, mitochondrial (Q04837) | 0 | 0 | 1.12 | 0.48 | ||
| AFG3-like protein 2 (Q9Y4W6) | 0 | 0 | 0.6 | 0 | ||
| Isoform 2 of Probable hydrolase PNKD (Q8N490) | 0 | 0 | 1.9 | 0.14 | ||
| Dihydrolipoyl dehydrogenase (E9PEX6) | 0 | 0 | 0.43 | 0 | ||
| Feline leukemia virus subgroup C receptor-related protein 1 (Q9Y5Y0) | 0.05 | 0 | 0.48 | 0.26 | ||
| Clathrin heavy chain (A0A087WVQ6) | 0.02 | 0 | 0.41 | 0.25 | ||
| Staphylococcal nuclease domain-containing protein 1(Q7K2F4) | 0 | 0 | 0.8 | 1.2 | ||
| Peptide-prolyl cic-trans isomerase FKBP8 (A0A0A0MTJ1) | 0 | 0 | 1.23 | 0.28 | ||
| Isoform 3 of cold shock domain-containing protein E1 (O75734) | 0 | 0 | 0.17 | 0 | ||
| 40S ribosomal protein S3 (P23396) | 12.4 | 6 | 1 | 3.7 | ||
| Electron transfer flavoprotein subunit alpha, mitochondrial (H0YK49) | 0.30 | 0.08 | 0 | 0 | ||
| cDNA FLJ60124, highly similar to Mitochondrial dicarboxylate carrier (B4DLN1) | 1.31 | 0.1 | 1.7 | 0 | 0.54 | |
| Leucine-rich PPR motif-containing protein 59, mitochondrial (Q96AG4) | 1.3 | 0.3 | 0.13 | 0 | 0.04 | |
| Peroxiredoxin-5, mitochondrial (P30044) | 0.33 | 0 | 0 | 0 | ||
IAC of DmHsp22 and negative control with anti-Flag antibodies from 2 and 1 mg of S1300 g isolated from human HeLa cells are subjected to MS analysis. Proteins that were detected only in DmHsp22 expressing cells or that were detected with higher abundancy than the control were considered as specific interactors of DmHsp22. A total of 137 and 72 proteins were identified from two independent IACs and classified as mitochondrial partners of DmHsp22 using UniProt database bioinformatic resources. The highlighted proteins are the ATP synthase (complex V) subunits. Columns identified as DmHsp22-Flag and control show the emPAI index representing the abundance of detected proteins interacting with DmHsp22-Flag and control at Physiological condition. Heat shock +6 column shows the emPAI of detected proteins interacting with DmHsp22 following heat shock at 42°C for 1 hour and 6 hours recovery at 37°C.