#METABOLOMICS WORKBENCH tkhatib_20241107_122614 DATATRACK_ID:5356 STUDY_ID:ST003666 ANALYSIS_ID:AN006023 PROJECT_ID:PR002274 VERSION 1 CREATED_ON January 16, 2025, 11:47 am #PROJECT PR:PROJECT_TITLE TGFB1-mediated intercellular signaling fuels cooperative cellular invasion PR:PROJECT_TYPE Proteomics Mass Spectrometry on conditioned media PR:PROJECT_SUMMARY SaGA-derived (Khatib 2023, Konen 2017) leader and follower subpopulation PR:PROJECT_SUMMARY conditioned media was collected and sequenced via Mass spectrometry/proteomics PR:PROJECT_SUMMARY to compare differences in cell secretion from subpopulations originally isolated PR:PROJECT_SUMMARY from the same parental population. PR:INSTITUTE Emory University PR:DEPARTMENT Hematology and Oncology PR:LABORATORY Marcus Lab PR:LAST_NAME Khatib PR:FIRST_NAME Tala PR:ADDRESS 201 Dowman Dr, Atlanta, GA 30322 PR:EMAIL tkhatib@emory.edu PR:PHONE 9102622080 PR:FUNDING_SOURCE This work was supported by NIH NCI grants R01CA250422 (A.I.M.), R01CA247367 PR:FUNDING_SOURCE (A.I.M.), R01CA236369 (A.I.M.), PO1CA257906 (A.I.M.), R50CA265345 (J.K.M.), PR:FUNDING_SOURCE National Science Foundation Graduate Research Fellowship (NSF-GRFP 1937971), and PR:FUNDING_SOURCE an Imagine, Innovate, Impact (I3) Research award from the Emory School of PR:FUNDING_SOURCE Medicine through the Georgia CTSA NIH award UL1-TR002378 (A.I.M. and H.G.). PR:CONTRIBUTORS Janna Mouw, Tala Khatib #STUDY ST:STUDY_TITLE TGFB1-mediated intercellular signaling fuels cooperative cellular invasion ST:STUDY_SUMMARY Conditioned media from SaGA-derived leader and follower subpopulations (Khatib ST:STUDY_SUMMARY 2023, Konen 2017) were collected and analyzed using mass spectrometry-based ST:STUDY_SUMMARY proteomics to assess differences in cell secretion profiles. Label-free ST:STUDY_SUMMARY quantification was employed to compare spectra against the 2020 human ST:STUDY_SUMMARY UniProtKB/Swiss-Prot database (20,379 target sequences). This analysis ST:STUDY_SUMMARY identified 1,174 proteins. Our findings revealed that the follower subpopulation ST:STUDY_SUMMARY produces and secretes TGFB1 and TGFB-related factors. Notably, the leader ST:STUDY_SUMMARY subpopulation secretes FN1 and ALCAM, which are downstream effectors of ST:STUDY_SUMMARY TGFB-mediated signaling. ST:INSTITUTE Emory University ST:LAST_NAME Khatib ST:FIRST_NAME Tala ST:ADDRESS 201 Dowman Dr, Atlanta, GA 30322 ST:EMAIL tkhatib@emory.edu ST:PHONE 9102622080 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENOTYPE_STRAIN H1299 cell line SU:AGE_OR_AGE_RANGE 45 SU:GENDER Male SU:CELL_STRAIN_DETAILS H1299 cell line SU:CELL_PASSAGE_NUMBER 3,4,5 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - Follower_1 Type:Conditioned media | Sample source:H1299 Lung cancer cell line RAW_FILE_NAME(Raw file name)=Follower_1.raw SUBJECT_SAMPLE_FACTORS - Follower_2 Type:Conditioned media | Sample source:H1299 Lung cancer cell line RAW_FILE_NAME(Raw file name)=Follower_2.raw SUBJECT_SAMPLE_FACTORS - Follower_3 Type:Conditioned media | Sample source:H1299 Lung cancer cell line RAW_FILE_NAME(Raw file name)=Follower_3.raw SUBJECT_SAMPLE_FACTORS - Follower_4 Type:Conditioned media | Sample source:H1299 Lung cancer cell line RAW_FILE_NAME(Raw file name)=Follower_4.raw SUBJECT_SAMPLE_FACTORS - Leader_1 Type:Conditioned media | Sample source:H1299 Lung cancer cell line RAW_FILE_NAME(Raw file name)=Leader_1.raw SUBJECT_SAMPLE_FACTORS - Leader_2 Type:Conditioned media | Sample source:H1299 Lung cancer cell line RAW_FILE_NAME(Raw file name)=Leader_2.raw SUBJECT_SAMPLE_FACTORS - Leader_3 Type:Conditioned media | Sample source:H1299 Lung cancer cell line RAW_FILE_NAME(Raw file name)=Leader_3.raw SUBJECT_SAMPLE_FACTORS - Leader_4 Type:Conditioned media | Sample source:H1299 Lung cancer cell line RAW_FILE_NAME(Raw file name)=Leader_4.raw #COLLECTION CO:COLLECTION_SUMMARY Cells were initially cultured in standard media for 24 h and then replaced with CO:COLLECTION_SUMMARY defined media after two rinses with 1X PBS. After 48 h, the media was collected CO:COLLECTION_SUMMARY and centrifuged at 250 g for 5 min to remove any detached cells, followed by a CO:COLLECTION_SUMMARY 20 min centrifugation at 2000 g to eliminate apoptotic bodies. The media was CO:COLLECTION_SUMMARY then concentrated using the Amicon ultra-15 centrifugal filter unit with a 3-kDA CO:COLLECTION_SUMMARY cut off and centrifugation at 4000 g at 4℃ for a final volume of 250 uL. CO:COLLECTION_SUMMARY Samples were flash-frozen before storage at -80℃. CO:SAMPLE_TYPE Lung #TREATMENT TR:TREATMENT_SUMMARY LC-MS/MS was performed on the secreted media content of adherent leader and TR:TREATMENT_SUMMARY follower cell populations in 2D culture. Cells were initially cultured in TR:TREATMENT_SUMMARY standard media for 24 h and then replaced with defined media [1X TR:TREATMENT_SUMMARY insulin-transferrin-selenium-ethanolamine, 0.5 mg/mL hydrocortisone, 1 ng/mL TR:TREATMENT_SUMMARY cholera toxin, 50 nM O-phosphorylethanolamine, and 5 nM triiodothyronine] after TR:TREATMENT_SUMMARY two rinses with 1X PBS. After 48 h, the media was collected and concentrated for TR:TREATMENT_SUMMARY LC-MS/MS. TR:TREATMENT_PROTOCOL_FILENAME Protocol #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Protein normalization, digestion, and LC-MS/MS data acquisition and analysis SP:SAMPLEPREP_SUMMARY were conducted at the Emory Integrated Proteomics Core. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters NanoAcquity CH:COLUMN_NAME Waters ACQUITY UPLC CSH C18 (150 x 2.1mm,1.7um) CH:SOLVENT_A 99.9% water; 0.1% formic acid CH:SOLVENT_B 99.9% acetonitrile; 0.1% formic acid CH:FLOW_GRADIENT 60 min gradient CH:FLOW_RATE 1.25 uL/min CH:COLUMN_TEMPERATURE 60 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Orbitrap Fusion Lumos Tribrid MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The data acquisition by LC-MS/MS protocol was adapted from a published procedure MS:MS_COMMENTS (Seyfried, Dammer et al. 2017) and was performed by the Integrated Proteomics MS:MS_COMMENTS Core Facility at Emory University. Derived peptides were resuspended in loading MS:MS_COMMENTS buffer (0.1% trifluoroacetic acid). Peptide mixtures were separated on a MS:MS_COMMENTS self-packed 1.7 µm CSH waters resin column (15 cm x 100 µm internal diameter MS:MS_COMMENTS (ID); New Objective, Woburn, MA) attached to a Dionex 3000 RSLCnano system and MS:MS_COMMENTS were monitored on a Orbitrap Fusion Lumos Mass Spectrometer (ThermoFisher MS:MS_COMMENTS Scientific, San Jose, CA). Elution was performed over a 60 min gradient at a MS:MS_COMMENTS rate of 1.25 µL/min (buffer A: 0.1% formic acid in water, buffer B: 0.1 % MS:MS_COMMENTS formic acid in acetonitrile): The gradient started with 1% buffer B and went to MS:MS_COMMENTS 5% in 6 sec followed by 30% in 53 minutes, and then increased to 99% within 1 MS:MS_COMMENTS minute and staying at 99% for another 4 minutes before ending at 1% for 2 MS:MS_COMMENTS minutes. The mass spectrometer was operated in data dependent mode in top speed MS:MS_COMMENTS mode with a cycle time of 3 seconds. Survey scans were collected in the Orbitrap MS:MS_COMMENTS with a 120,000 resolution, 400 to 1600 m/z range, 400,000 automatic gain control MS:MS_COMMENTS (AGC), 246 ms max injection time and rf lens at 30%. Higher energy collision MS:MS_COMMENTS dissociation (HCD) tandem mass spectra were collected in the Orbitrap with a MS:MS_COMMENTS 15,000 resolution, collision energy of 35%, an isolation width of 1.6 m/z, AGC MS:MS_COMMENTS target of 100000, and a max injection time of 54 ms. Dynamic exclusion was set MS:MS_COMMENTS to 30 seconds with a 10 ppm mass tolerance window. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS LQF Intensity MS_METABOLITE_DATA_START Samples Follower_1 Leader_1 Follower_2 Leader_2 Follower_3 Leader_3 Follower_4 Leader_4 Factors Type:Conditioned media | Sample source:H1299 Lung cancer cell line Type:Conditioned media | Sample source:H1299 Lung cancer cell line Type:Conditioned media | Sample source:H1299 Lung cancer cell line Type:Conditioned media | Sample source:H1299 Lung cancer cell line Type:Conditioned media | Sample source:H1299 Lung cancer cell line Type:Conditioned media | Sample source:H1299 Lung cancer cell line Type:Conditioned media | Sample source:H1299 Lung cancer cell line Type:Conditioned media | Sample source:H1299 Lung cancer cell line A0A0C4DH29 0 47206000 43911000 79583000 0 73902000 0 66263000 A4UGR9 20298000 26899000 0 0 0 0 0 0 A6H8Y1 0 0 2431500 83660000 2569800 2222700 0 7281800 A6NCE7;Q9GZQ8;Q9H492 13578000 21284000 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REV__Q9Y5E7 19618000 29928000 23604000 22017000 26054000 27909000 19291000 29969000 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name A0A0C4DH29 A4UGR9 A6H8Y1 A6NCE7;Q9GZQ8;Q9H492 A6NDG6 Q6S5H5;A6NI47;Q6S545 A8MPP1;Q92771 P62308;A8MWD9 CON__P01030;CON__ENSEMBL:ENSBTAP00000007350 CON__ENSEMBL:ENSBTAP00000018229 CON__ENSEMBL:ENSBTAP00000024146 CON__ENSEMBL:ENSBTAP00000038329 CON__P00735 CON__P00761 CON__P00978 CON__P01044-1;CON__Q2KJ62;CON__P01045-1 CON__P01966 CON__P02533;P02533;CON__Q04695;CON__Q9QWL7;CON__Q6IFX2;CON__P19012;CON__P08779;Q04695;P19012;P08779 CON__P02768-1;P02768 CON__P02769 CON__P05787;P05787;CON__H-INV:HIT000292931 CON__P06868 CON__P12763 CON__P13645;P13645 CON__P13647;P13647 CON__P15497 CON__P15636 CON__P17690 CON__P34955 CON__P35527;P35527 CON__P35908;CON__P35908v2;P35908 CON__P81644 CON__Q29443;CON__Q0IIK2 CON__Q0V8M9 CON__Q2KJF1 CON__Q2UVX4 CON__Q3KUS7 CON__Q3MHN5 CON__Q3SX09 CON__Q3SX28 CON__Q3SZ57 CON__Q3SZH5 CON__Q3SZR3 CON__Q3SZV7 CON__Q3T052 CON__Q3TTY5 CON__Q58D62 CON__Q9TRI1 CON__REFSEQ:XP_001252647;CON__A2I7N3 E9PAV3;Q13765 G2XKQ0;P63165 O00115 O00142 O00148 O00154 O00159 O00161 O00170 O00193 O00203 O00231 O00232 O00233 O00244 O00267 O00273 O00299 O00303 O00339 O00391 O00410 O00429 O00461 O00462 O00468 O00469 O00487 O00505 O00560 O00567 O00571;O15523 O00584 O00622 O00625 O00629 O00754 O00764 O14530 O14561 O14579 P47813;O14602 O14607 O14618 O14657 O14672 O14737 O14744 O14745 O14773 O14786 O14818 O14879 O14907 O14908 O14929 P19105;O14950 O14964 O14974 O14979 O14980 O15031 O15041 O15066 O15067 O15072 O15078 O15116 O15143 O15144 O15145 O15198 O15212 O15230 O15240 O15347 O15355 O15371 O15372 O15400 O15427 O15446 O15498 O15511 O15514 O43143 O43175 O43237 O43240 O43242 O43252 O43324 O43390 O43395 O43396 O43399 O43405 O43488 O43491 O43493 O43504 O43505 O43556 O43583 O43592 O43598 O43670 O43681 O43684 O43707 O43747 O43765 O43768 O43776 O43809 O43813 O43818 O43847 O43852 O43924 O60216 O60245 O60256;Q14558 O60271 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P36551 P36578 P36639 P36871 P36873 P36915 P36955 P36957 P36969 P37108 P37198 P37235 P37802 P37837 P38159;Q96E39;O75526 P38571 P38606 P38646 P38919 P39019 P39023 P39060 P39687 P39748 P40123 P40189 P40222 P40227 P40429;Q6NVV1 P40925 P40926 P41091;Q2VIR3 P41208 P41219 P41227 P41250 P41252 P41271 P41567;O60739 P42126 P42166 P42167 P42285 P42566 P42568 P42677 P42704 P42785 P42892 P43034 P43146 P43243 P43358 P43487 P43490 P43686 P45877 P45973 P45974 P46060 P46063 P46087 P46108 P46109 P46379 P46459 P46776 P46777 P46778 P46779 P46781 P46782 P46783;Q9NQ39 P46821 P46926 P46940 P46976 P47755 P47756 P47897 P47914 P48147 P48307 P48444 P48507 P48509 P48634 P48637 P48643 P48723 P48739 P48960 P49006 P49023 P49189 P49257 P49321 P49327 P49368 P49411 P49419 P49458 P49459 P49588 P49589 P49591 P49593 P49720 P49721 P49736 P49746 Q86TX2;P49753 P49756 P49767 P49773 P49903 P49915 P50395 P50454 P50502;Q8IZP2;Q8NFI4 P50552 P50579 P50583 P50895 P50897 P50914 P50990 P50991 P51003 P51114 P51149 P51531;P51532 P51570 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