EFFECTS OF HEMATURIA ON THE PROTEOMIC PROFILE OF URINARY

Transcription

1 Supporting Information EFFECTS OF HEMATURIA ON THE PROTEOMIC PROFILE OF URINARY EXTRACELLULAR VESICLES: TECHNICAL CHALLENGES. #Francesca Raimondo 1*, #Clizia Chinello 1, Martina Stella 1, Lucia Santorelli 1, Fulvio Magni 1 and Marina Pitto 1. 1 Department of Medicine and Surgery, University Milan Bicocca, Monza, Italy Figure S1. Effect of the blood contamination on Urine Extracellular vesicle (UEv) protein profile and content. UEv proteins were separated by NuPAGE electrophoresis system and stained by Coomassie Blue. Densitometric analysis was performed by ImageQuant TL software (GE Healthcare). A) UEv protein profiles obtained by 7 different control urine pools (A-G) with/out blood; the lanes are loaded with UEv proteins coming from the same amount of starting urine (3 ml), in order to check the different UEv recovery. B) Densitometric analysis. Total proteins: optic density (OD) of the total lanes corresponding to the protein profiles of UEv isolated from urine with increasing levels of blood (0, 1+, 2+, 3+, 4+). We considered sample without blood contamination (0) as 100%. Uromodulin (UMOD): UMOD was evaluated by OD of the band at kda. We considered sample without blood contamination (0) as 100%. Hemoglobin (Hb): Hb enrichment was reported as the OD of the band observed at the same height of the Hb standard loaded on the same gel. In this case, we considered sample without higher contamination (4+) as 100%. The results are representative of four independent experiments and are expressed as percentage average + absolute error, calculated considering the error propagation. UMOD, Uromodulin; Hb, Hemoglobin; MW, Molecular Weight marker. Figure S2. Effect of trypsin treatment on the enrichment of the exosome markers. Comparison of fractions obtained from urine without blood contamination and with high level of hematuria (3+ and 4+). A) Protein profiles by Rosso Ponceau Staining after western blotting. +). B) Immunoblotting with antibodies against exosomal markers (TSG-101 and AQP2). Proteins coming from the same amount of starting urine for each urine subfraction type were loaded, in order to check the different protein recovery. P1, sediment; P2, large membrane fragments and large vesicles (protein vesicles corresponding to 1 ml of urine); UEv, exosome enriched fraction (protein vesicles corresponding to 7 ml); U, urine proteins after sediment removal (soluble proteins corresponding to 0.2 ml); Sn, supernatant after the xg ultracentrifugation (soluble proteins corresponding to 0.2 ml). We loaded in the gel protein amounts derived from different volumes to avoid overloading of the lanes, because the yield of protein from each subfraction type is very different: Sn >> P2 >> UEv. The S1

2 nitrocellulose filters probed with the same antibody were incubated with the same ECL substrate and the images were acquired by a CCD camera with the same exposition time. Figure S3. Subcellular localization of proteins identified in UEv. Figure S4: Venn diagram describing the overlapping and unique proteins in the UEv samples after trypsin treatment with and without blood. Table S1. List of all the identified proteins in UEv isolated from urine contaminated with blood or not, after trypsin treatment: protein description, subcellular localization, tissue specificity and presence in other protein datasets. Table S2. List of proteins identified in each sample of UEv obtained from urine blood-contaminated or not, submitted to trypsin treatment or not: proteins identified using Mascot search. Table S3. List of proteins identified in each sample of UEv obtained from urine blood-contaminated or not after trypsin treatment: proteins identified through Mascot via Progenesis QI. Table S4. Number and tissue specificity of identified proteins in each sample, contaminated or not with blood after trypsin treatment. Table S5. Label-free quantitative analysis data. Table S6. Distribution of up, down and unchanged proteins in relation to the blood contamination levels. S2

3 Figure S1. Effect of the blood contamination on Urine Extracellular vesicle (UEv) protein profile and content. UEv proteins were separated by NuPAGE electrophoresis system and stained by Coomassie Blue. Densitometric analysis was performed by ImageQuant TL software (GE Healthcare). A) UEv protein profiles obtained by 7 different control urine pools (A-G) with/out blood; the lanes are loaded with UEv proteins coming from the same amount of starting urine (3 ml), in order to check the different UEv recovery. B) Densitometric analysis. Total proteins: optic density (OD) of the total lanes corresponding to the protein profiles of UEv isolated from urine with increasing levels of blood (0, 1+, 2+, 3+, 4+). We considered sample without blood contamination (0) as 100%. Uromodulin (UMOD): UMOD was evaluated by OD of the band at kda. We considered sample without blood contamination (0) as 100%. Hemoglobin (Hb): Hb enrichment was reported as the OD of the band observed at the same height of the Hb standard loaded on the same gel. In this case, we considered sample without higher contamination (4+) as 100%. The results are representative of four independent experiments and are expressed as percentage average + absolute error, calculated considering the error propagation. UMOD, Uromodulin; Hb, Hemoglobin; MW, Molecular Weight marker. S3

4 Figure S2. Effect of trypsin treatment on the enrichment of the exosome markers. Comparison of fractions obtained from urine without blood contamination and with high level of hematuria (3+ and 4+). A) Protein profiles by Rosso Ponceau Staining after western blotting. +). B) Immunoblotting with antibodies against exosomal markers (TSG-101 and AQP2). Proteins coming from the same amount of starting urine for each urine subfraction type were loaded, in order to check the different protein recovery. P1, sediment; P2, large membrane fragments and large vesicles (protein vesicles corresponding to 1 ml of urine); UEv, exosome enriched fraction (protein vesicles corresponding to 7 ml); U, urine proteins after sediment removal (soluble proteins corresponding to 0.2 ml); Sn, supernatant after the xg ultracentrifugation (soluble proteins corresponding to 0.2 ml). We loaded in the gel protein amounts derived from different volumes to avoid overloading of the lanes, because the yield of protein from each subfraction type is very different: Sn >> P2 >> UEv. The nitrocellulose filters probed with the same antibody were incubated with the same ECL substrate and the images were acquired by a CCD camera with the same exposition time. S4

5 Figure S3. Subcellular localization of proteins identified in UEv. Figure S4: Venn diagram describing the overlapping and unique proteins in the UEv samples after trypsin treatment with and without blood. S5

6 Table S4. Number and tissue specificity of identified proteins in each sample, contaminated or not with blood after trypsin treatment. Hematuria level (Combur Test) Identified proteins (N) Unique proteins (N) Tissue Specificity Ubiquitous/mixed 49% 49% 48% 50% 50% Bone Marrow, Immune System 5% 4% 4% 4% 5% Immunoglobulins 5% 4% 5% 5% 6% Liver 8% 9% 9% 10% 10% Urinary tract 9% 9% 9% 8% 9% Others 24% 24% 24% 23% 20% Note: 0, without blood contamination; 1+, 2+, 3+, 4+ describe the hematuria level of the urine assessed by Combur test (Roche); N, number. Table S6. Distribution of up, down and unchanged proteins in relation to the blood contamination levels. Hematuria Ratio Protein number (%) 0,5 0 (0%) 0 (0%) 0 (0%) 1 (~0%) 0,5 < R < (99%) 139 (97%) 129 (92%) 119 (84%) 2 2 (1%) 4 (3%) 14 (8%) 23 (16%) Note: control UEv, in the absence of blood addition is set as one. S6