A Conventional This study B One glomerulus One glomerulus

Lysis in 8M Urea Lysis in 4% SDS SDS

Isolation by magnetic beads SDS

Buffer exchange

Digestion Digestion Try LysC pH>7

C18 Stage tip Buffer exchange

pH<4

functionalized nLC-MS/MS nLC-MS/MS magnetic beads protein/peptide C 10000 9000 8000 7000 SP3 6000 (ultrasensitive sample prep.) 5000 Stagetips 4000 3000 Identified peptides 2000 1000 0 0 1 2 8 16 32 ~100 Mouse glomeruli

Suppl. Fig. 1 S1. Sample preparation methods. A. Scheme of conventional (C18 Stage Tip) and ultrasensitive proteomic sample preparation methods. B. Manual microdissection and isolation of single glomeruli. The arrow indicates a single isolated glomerulus subjected to proteome analysis. The tip capacity is 10µl. C. Stagetips, a conventional proteomic sample preparation method, is compared with ultrasensitive (SP3) proteome analysis. The indicated amounts of mouse glomeruli were prepared by the respective sample preparation protocols, and peptide numbers after nLC-MS/MS are plotted (all FDR<0.01).

A Suppl. Fig.2 C E

log10(iBAQ) Intensity Intensity

10 Number20 30 of peptides40 50 60 1 1 2 4 6 8 2 4 6 8 4 5 6 7 8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Co n 1 tr 0 o Human proximaltubule l p m tu r (S1) o Rank protein o b 2 x u 0 u i m s l e e T a i l me m m Loading... 3

0 o (mi T u A s n L e ) 4 0 m 1 0 CD Tubule o Tubule u s e 5 0 p h (S1) tu r o u b x m u i m a 6 l e 1000 n 0 a l D B F

log (iBAQ), human proximal tubule log10(iBAQ) 5 8 7

10 6 4 5 6 7 8 0 Component 2 (22.5%) log Mouse proximaltubule 5 -20 0 20 40 10 -40 SLC5A1 S1 (iBAQ), mouseproximal tubule S2 proximal tubule SLC5A10 Rank protein SLC5A12 6 -20 CUBN SLC16A1 Loading... SLC25A12 SLC3A2 Component 2(47.1%) S3 SLC3A1 7 0 SLC5A2 Loading... SLC22A6 SLC25A5 SLC25A13 CLTC LRP2 SLC25A10 R=0.7 SLC9A3R1 SLC27A2 SLC25A3 20 8 SLC25A4 ATP1A1 ATP1B1 1500 CCD TAL control ACTB 40 S2. Overview of single tubule proteomics results. A. Total ion current during LC-MS/MS acquisition of samples from 0 (vehicle solution only) and 1 mouse tubule (S1). B. PCA of global proteomic dataset from single segments dissected from a Wildtype mouse. C. Peptide numbers from single tubule samples from two different mice. Data from mouse (14 S1 tubules, 4 TAL tubules, 4 CCD) and human (6 S1 tubules) are shown. D. Dynamic range of proteome in single mouse tubules. 1500 proteins could be quantified in more than 4 single tubules. iBAQ is a parameter quantifying absolute protein abundance (corresponding to copy numbers). E. Dynamic range of proteome of single human proximal tubules. Approximately 1000 proteins could be quantified. F. Comparison of absolute protein abundance in single mouse and human tubules. The correlation is significant (R= 0.7). Correlation coefficients observed within one species were R>0.8, even from different animals.

A WT1het Wildtype

B C Wildtype WT1het 2

1 WT1hetWildtypeWT1hetWildtype Loading... 0 Lamp1 expression R= 0.71 -70kDa -1 0 1 2 Lama5 expression -55kDa 2 -35kDa 1

-25kDa Loading... 0 Lamp1 expression -1 R= -0.72 -3 -2 -1 0 1 2 Actn1/4 expression Suppl. Fig. 3 S3. Morphology, functional parameters and targeted proteomics of Wt1 heterozygous knockout mice. A. PAS staining demonstrates glomerular damage and partial sclerosis in 14 week old Wt1het knockout mice and control mice. Scale bar = 100 µm. B. Coomassie gel demonstrating proteinuria in Wt1het mice. C. Examples of correlation of normalized protein expression levels from single glomerular proteome data. Each dot represents measurements of expression level (log2 scale) from 1 glomerulus, which is color-coded according to genotype. Normalized expression of different, exemplary proteins is plotted vs each other and unravels strong positive and significant correlation of laminin alpha 5 (LAMA5) and LAMP1, but negative-correlation between ACTN1/4 and LAMP1. No correlation could be determined between nephrin and ACTN1/4.

A Slc22a30 Slc3a1 Slc5a10 Slc22a6 Phb Vps29 Atp5a1 Myl6 H2afy Sdhb Rab11b Vps35 Gnas Tubb4b Pcx Fbp1 Slc13a3 Gnai1 Lrp2 Rac1 Prkar2a Myh9 Atp1b1 Cdh16 Gpi Hk1 Slc43a2 Slc7a8 Slc3a2 Slc5a2 Slc5a12 Slc16a1 Ca2 Slc26a1 Slc22a8 Pdzk1ip1 Atp6v1a Slc2a2 Slc6a19 Cubn Slc4a4 Idh1 Pdzk1 Slc22a12 Psma6 Slc34a1 Slc22a5 Slc17a3 Aqp1 Slc27a2 Psmb5 Slc22a18 G6pc Alb Col4a2 Slc22a1 Ca3 Slc9a3 Atp1a1 Sdha Hist1h2bc Rhoa Ptgr2 Slc33a1 Rab1A Lamp1 Slc6a20b Lamp2 Slc22a2 Pck1 Ldha Cltc Slc9a3r1 Gapdh S3 S1 RNAseqS2

Slc22a30 Slc3a1 Slc5a10 Slc22a6 Phb Vps29 Atp5a1 Myl6 H2afy Sdhb Rab11b Vps35 Gnas Tubb4b Pcx Fbp1 Slc13a3 Gnai1 Lrp2 Rac1 Prkar2a Myh9 Atp1b1 Cdh16 Gpi Hk1 Slc43a2 Slc7a8 Slc3a2 Slc5a2 Slc5a12 Slc16a1 Ca2 Slc26a1 Slc22a8 Pdzk1ip1 Atp6v1a Slc2a2 Slc6a19 Cubn Slc4a4 Idh1 Pdzk1 Slc22a12 Psma6 Slc34a1 Slc22a5 Slc17a3 Aqp1 Slc27a2 Psmb5 Slc22a18 G6pc Alb Col4a2 Slc22a1 Ca3 Slc9a3 Atp1a1 Sdha Hist1h2bc Rhoa Ptgr2 Slc33a1 Rab1A Lamp1 Slc6a20b Lamp2 Slc22a2 Pck1 Ldha Cltc Slc9a3r1 Gapdh

Pearson‘s R normalized expr. Amino acid transporter decreased -1 +1 -4.5 +4.5 increased Albumin Col IV B

50 Wildtype e

n WT1het i n

i 40 t a e r c

l 30 o m m / l 20 o m µ 10 urinary amino acid concentration, 0 p r r n n y it l s t e u r e s g s h e s l l la a y e Il e y h i r A T S A G G A C V C M L T P H A

Suppl. Fig. 4 S4. Analysis of single tubule proteome and function. A. Correlational matrix of protein expression in single tubules. Proteins with amino acid transporter activity and collagen/albumin are marked. Pearson Correlation coefficient across all proteins and all tubules is clustered according to Euclidean distance. The normalized mRNA expression levels in S1, S2 and S3 proximal tubules (according to Lee, Chou and Knepper, JASN 2015) are depicted for every individual protein. B. Amino acid quantification in WT1het mice and control mice. Urinary concentrations were determined and normalized by creatinine. All changes of the amino acids depicted here were not significant (two-tailed t-test, significance designated as p < 0.05). n = 6 /group. Asp: Aspartic acid, Thr: Threonine, Ser: Serine, Asn: Asparagine, Gln: Glutamine, Gly: Glycine, Ala: Alanine, Cit: Citrulline, Val: Valine, Cys: Cysteine, Met: Methionine, Ile: Isoleucine, Leu: Leucine, Tyr: Tyrosine, Phe: Phenylalanine, His: Histidine, Arg: Arginine.

A vehicle Doxorubicin

B

1500

1000

(mg/g x 0.1) 500 Albumin/Creatinine ratio

0 veh Doxorubicin Suppl. Fig. 5 S5. Morphology and proteinuria of doxorubicin treated mice. A. Representative PAS staining of kidneys from doxorubicin and vehicle treated (control) glomeruli. Scale bar = 100 µm. B. Albuminuria in control and doxorubicin treated mice from all three mice analyzed in this study.

A

60

50 n.s.

40

30 1.0 0.8 0.6 Ratio (g/g x 0.1)

Albumin-Creatinine 0.4 0.2 0.0 T ) O O O 7 W K K K y L Z a B . . d t. t t ( a a a S C C C T N + T W

B veh. NTS W ildtype Cat. B KO KO Cat. L Cat. Z KO

Suppl. Fig. 6 S6. Morphology and proteinuria of cathepsin B/L/Z knockout mice. A. Proteinuria measured in mice of control and cathepsin knockout mice. N.s. = not significant in a two- tailed t-test. B. Representative PAS staining of kidneys demonstrating morphology of cathepsin B/L/Z KO and control mice with nephrotoxic serum (NTS) and vehicle treatment. Scale bar = 100µm.

NPHS1 Patient 1 NPHS1 Patient 2

control Patient 1 control Patient 2

Suppl. Fig. 7 S7. Histology of congenital nephrotic syndrome patients and controls in the study. PAS staining of the four patients analyzed in the study.

A B C 8 8 7 7 8 6 6 5 5 6 SCARB2 4 Loading... Loading... Loading... 4 3 4 -log(p-value) -log(p-value) -log(p-value) 3

LAMP1 2 2 2 1 1 0 0 0 -8 -4 0 4 8 -4 -3 -2 -1 0 1 2 3 4 -4 -3 -2 -1 0 1 2 3 4 log2(FSGS/con) log2(NPHS1/con) log2(NPHS1 patient/control)

Suppl. Fig. 8 S8. GO Term analysis of proteinuric vs non-proteinuric glomeruli. Volcano plots corresponding to Fig. 3 A-C are depicted. Blue dots represent mitochondrial protein (GO- CC), which are the most consistently decreased proteins in the dataset.

A

Cut slices (cryosections) Consecutive slides (cryosections)

1 2

3 4

B C Glomerular proteins Tubular proteins Slice Slice 1 2 3 4 1 2 3 4 ACTB MYH9 * CA2 ACTN4 CUBN MYO1E * NPHS2 * LRP2 LAMP2 * ATP6V1B2 COL4A5 CTSB * SLC27A2 ARHGDIA ATP6V1A CTSZ * COL4A4 SLC5A12 ACTN4 SLC3A1 CTSD COL4A2 * SLC25A3 COL4A3 * COL18A1 AQP1 CANX SLC3A2 ACTA1 SYNPO ATP6V1C1 COL3A1 * SLC22A2 LAMP1 MYLK ATP6V1H CTTN SLC44A2 ACTN1 COL4A1 * SLC25A24 LAMA5 SLC4A4 COL1A1 COL1A2 SLC25A1

log2(intensity) 22 32 not id.

Suppl. Fig. 9 S9. Proteomic analysis of laser caption microdissection. A. In ~10 µm-thick kidney sections, glomerular areas were cut by laser-dissection, and consecutive slides (1 µm thickness) were stained with PAS. B. Glomerular proteins discovered from a single glomerular section involve basement membrane proteins as well as several podocyte- specific proteins involved in human disease (marked with a *). Only selected proteins from the entire dataset (more than 1200 proteins) are depicted. C. Tubular proteins were not found in the glomerular sections, except in section 4 where tubular tissue was arbitrarily laser-dissected. Representation of selected proteins.