Supporting InformationGlasgow et al. 10.1073/pnas.1611642113SI Materials and MethodsMS of Photooxidation with Streptavidin–MB. We combined 10 μMstreptavidin in PBS with MB–biotin (AttoTec) at 14 μM or PBS asa control. Samples were photooxidized for 0, 3, 10, 30, and 90 s ona solar simulator with 615/40 nm light. A concentration of 0.4%Rapigest and TNE buffer (50 mM Tris·HCl pH 8, 100 mM NaCl,1 mM EDTA) was added to samples, and solutions were trypsin-digested overnight at 37 °C. LC–MS/MS was performed as de-scribed in Ligand-Proximal Photooxidation, LC–MS/MS, exceptMS database contained sequences only for streptavidin, avidin,and trypsin. The areas of the modified and unmodified peaks weremanually integrated.

Imaging of Peptide Binding on Tissue Sections. NP41 conjugated toMB, DBF, or biotin was topically applied to frozen, unfixed mousefacial nerve sections (10 mm) at 375 mM in 0.5×HBSS for 20 minand then washed in PBS. For imaging biotin–NP41, sections wereincubated with an avidin-conjugated peroxidase (ABC Reagent,Vector Labs) and stained for DAB precipitation. Slides wereimaged with white light microscopy. Confocal microscopy wasused to image NP41–MB (640 nm excitation, 670 nm emission)and DBF–NP41 (488 nm excitation, 520 nm emission; Nikon).

Imaging NP41–SOGs in Vivo. Adult SKH1 mice (Charles RiverLaboratories) or wild-type albino C57BL6 mice (Jackson Labo-ratories) were used for all experiments, and injections were donei.v. through the tail vein.We injected 300 nmol NP41–DBF and imaged it after 3 h of

washout. We injected 150 nmol eosin–NP41 and imaged it 2 hpostinjection. We injected 200 nmol MB-NP41 and imaged it 2.3 hpostinjection. For imaging, mice were anesthetized with an i.p.injection of 100 μL of a 1:1 mixture of ketamine HCl (100 mg/mL)and midazolam (5 mg/mL), and sciatic nerves were exposed andimaged with Zeiss Lumar.

Ligand-Proximal Photooxidation, LC–MS/MS. Homogenates were sol-ubilized in four volumes of freshly prepared buffer containing15 mM Na2HPO4, pH 7, 150 mM NaCl, 1 mM EDTA, 0.2 mMAEBSF, 10 mg/mL aprotinin, 1% Nonidet P-40, 0.5% sodiumdeoxycholate, and 1 mM DTT. Solution was centrifuged for 5 minat 930 × g and decanted to monomeric avidin agarose beads(Pierce Ultralink) and incubated on ice for 1 h with mild stirring.Supernatant was removed and discarded, and beads were washedthree times on ice.The hydrazone bond was released with two additions of 200 mL

8MureaTris buffer, pH7, followed by 8Murea 0.6% trifluoroaceticacid (TFA), pH 3, then 50 mM triethylamine, each time trans-ferring the eluted protein to the top of aMillipore Ultracon 0.5 mLconcentrator tube. The Filter-Aided Sample Prep (FASP) methodwas used to remove detergent. Reduction of disulfide bonds duringFASP used 5 mM DTT (Sigma-Aldrich) followed by 12 mMiodoacetamide (Sigma-Aldrich). The buffer was exchanged to100 mM ammonium acetate before overnight trypsin (Promega,1 μg) digestion at 37 °C. The digest was acidified with 0.4% TFA,and then C18 reverse-phase solid-phase extraction was used toprepare samples of ∼20 μL for LC–MS (TopTip, Glygen Corp.).Two 3-h LC–MS runs were done for each of the four samples

(Orbitrap XL, Thermo Fisher). In each LC–MS run, an 8 μLsample was injected. Peptide samples were separated by reverse-phase chromatography on a HPLCy (HPLC) column (100 μminner diameter, Polymicro Technologies fused silica) that waspacked in-house with a 7-cm stationary phase (Zorbax SB-C18,

80 Å, 5 μm, Agilent) and connected to an Agilent 1100 HPLCand autosampler with a flow rate of 200 μL/min and a flow splitter(microTee, Upchurch). The HPLC was coupled to a LTQ-Orbi-trap XL mass spectrometer (Thermo Scientific) equipped with ananoelectrospray ion source (Thermo Scientific). Peptides wereloaded onto the column with 100% buffer A (100% H2O, 0.1%formic acid) and eluted at approximately 200 nL/min over a148-min linear gradient from 2% to 30% buffer B (10% H2O, 90%acetonitrile, 0.1% formic acid) [all concentration percentages ex-pressed as (vol/vol)]. After the gradient, the column was washedwith 100% buffer B and reequilibrated with buffer A. Massspectra were acquired in a data-dependent manner, with an au-tomatic switch between MS and MS/MS scans. High-resolutionMS scans were acquired in the Orbitrap (60,000 FWHM, targetvalue 106) to monitor peptide ions in the mass range of 400–1,600m/z, followed by collision-induced dissociation (CID) MS/MSscans in the ion trap (minimum signal threshold, 1,000; targetvalue, 105; isolation width, 3 m/z) of the 10 most intense precursorions. To avoid multiple scans of dominant ions, the precursor ionmasses of scanned ions were dynamically excluded from MS/MSanalysis for 52 s. Singly charged ions and ions with unassignedcharge states were excluded from MS/MS fragmentation.The mass spectra data were searched with Sequest (Bioworks

version 3.3.1 software, Thermo Fisher) using up to three modifi-cations per peptide—that is, +15.9949 Da for methionine andtryptophan and also +3.9949 and +31.9898 Da for tryptophan—todetect kynurenine reverted from the protein–hydrazone linkage.Probability values were calculated by the software, and a filter ofP ≤ 0.05 was applied to the peptide spectrum matches (PSMs) forinclusion as a hit. Proteins with fewer than four hits or PSMs werenot included in the results shown in the final ratio plots. Thedatabase was UniProtKB/Swiss-Prot release number 2015_02 ofMus musculus. Spectral counts for peptide–protein matches weretabulated using Matlab software (Mathworks). The output fromthe Bioworks Search Results File (.srf) was exported as an Excelcomma separated value (.csv) spreadsheet.The spreadsheet data, for each LC–MS run, were imported into

Matlab. A Matlab script was written in-house to collate the listednumber of spectral counts for each protein observed in each run.In Excel, the spectral count hits for each condition were totaled—light versus NL or L-peptide versus D-peptide control.To check the peptide probability calculation, the data were

searched against a reversed database and listed with the same P ≤0.05 filter as above. The number of False Positive Hits wassummed for all of the proteins. This result was divided into thetotal number of hits for the search of the forward database,multiplied by 100, to give a false discovery rate (FDR) at thepeptide level of 4.5%, in agreement with the filter that was set.The probability value P is an inexact estimate for FDR, andhigher values of P (for MS/MS spectra from peptides alreadyidentified in multiple datasets) were accepted to reduce vari-ability in the ratio of PO/(PO + NL).

Alternate Data Analysis of Ligand-Proximal Photooxidation LC–MS/MSMeasurements. LC–MS/MS raw files were converted to the openmzML format with msconvert. Proteowizard (40) mzML files weresearched by the Comet search engine (41) against UniProtKB/Swiss-Prot protein database version 57.15 of M. musculus concatenatedwith the sequences of common contaminants. Search parameters forthe peptide identification included a precursor mass tolerance of25 ppm, a minimum of one tryptic terminus, and a maximum oftwo internal trypsin cleavage sites. Cysteine carbamidomethylation

Glasgow et al. www.pnas.org/cgi/content/short/1611642113 1 of 8

(+57.021 Da) was set as a static amino acid modification, andmethionine and tryptophan oxidation (+15.995 Da), tryptophanoxidation to kynurenin (+3.995 Da), and tryptophan oxidationto formylkynurenine (+31.989 Da) were set as differentialmodifications. The PeptideProphet and ProteinProphet toolsof the Trans-Proteomic Pipeline (TPP version 4.5) (42) wereused for probability scoring of peptides and proteins, and identi-fications were filtered to an FDR of ≤1%. Identified peptides werequantified by label-free quantification using the Progenesis (Non-linear Dynamics) software package. Data normalization, imputa-tion for missing values, and statistical testing were carried out usingthe statistical package MSstats (version 2.1.5) (43) within the Renvironment.

In Vivo Photooxidation and Labeling with Intramolecular Pyridylhydrazineand Biotin for MS Identification. SKH1 was tail vein-injected with450 nmol NP41–DBF–HB or the D-amino acid peptide. After 4 hof washout, mice were anesthetized with an i.p. injection of100 mL of a 1:1 mixture of ketamine HCl (100 mg/mL) andmidazolam (5 mg/mL), sciatic nerves were exposed, and one sidewas photooxidized with 480 nm (30 nm bandwidth, 0.07 W/cm2)of light on a solar simulator for 15 min, whereas the other waskept dark. Nerves were harvested from killed animals and re-acted in 50 mM Mes buffer, pH 5.5. Tissue was processed asdescribed previously for LC–MS/MS analysis.

TRICEPS-Based NP41–FAM–HB Imaging. Sciatic nerves from miceinjected with NP41–FAM–HB were exposed to either 100 mMsodium periodate (NaIO4) for 1 h at 25 °C or water and thenwashed overnight at 4 °C before imaging FAM fluorescence asdescribed in Materials and Methods.

TRICEPS-Based LRC, Sample Preparation, LC–MS/MS, and Analysis.Nerves were dissociated by indirect sonication (100% ampli-tude, 0.8 cycle, 60 s) in a VialTweeter (Hielscher) in 2% (wt/vol)RapiGest surfactant (Waters). Proteins were reduced with 5 mMTCEP (Pierce) at 20 °C for 30 min and alkylated with 10 mMiodoacetamide (Fluka) at 20 °C for 30 min. Trypsin (Sigma-Aldrich) was added at a 1:20 ratio to protein and incubated at37 °C overnight. Samples were heated to 96 °C for 10 min toinactivate proteases and cleared by centrifugation (13,000 × g for10 min). Affinity purification was performed by addition of 80 μLwashed Streptavidin Plus UltraLink Resin (Pierce) and in-cubated for 2 h on a slow rotator at 4 °C. Beads were washedextensively in Mobicols (Boca Scientific) connected to a Vac-Man Laboratory Vacuum Manifold (Promega) with four sepa-rate buffers: 5 M NaCl; followed by 100 mM NaCl, 100 mMglycerol, 50 mM Tris, and 1% Triton-X 100; followed by 100 mMNaHCO3 pH 11; followed by 50 mM ammonium bicarbonate.Washed beads were incubated with 400 μL 50 mM ammoniumbicarbonate containing 2 μL PNGase F (New England Biolabs)in an end-over-end shaker overnight at 37 °C. Peptides wereeluted by spinning of the Mobicol and subsequent addition ofanother 500 μL of 50 mM ammonium bicarbonate. Combinedeluates were acidified with 40 μL 10% formic acid and subjectedto C18 purification using 3–30 μg UltraMicroSpin Columns (TheNest Group) according to the manufacturer’s instructions.Peptide samples were separated by reversed-phase chroma-

tography on a high-performance liquid chromatography column(75 μm inner diameter; New Objective) that was packed in-housewith a 10-cm stationary phase (Magic C18AQ, 200 Å, 3 μm,Michrom Bioresources) and connected to a nano-flow EASY-nLC II Liquid Chromatograph (Thermo Scientific).The nLC system was coupled to an LTQ-Orbitrap XL mass

spectrometer (Thermo Scientific) equipped with a nanoelectrosprayion source (Thermo Scientific). Peptides were loaded onto thecolumnwith 95%buffer A (98%water, 2%acetonitrile, 0.1% formicacid) and eluted with 300 nL/min over a 40 min linear gradient from

5% to 35% buffer B (2% water, 98% acetonitrile, 0.1% formic acid)[all concentration percentages expressed as (vol/vol)]. After thegradient, the column was washed with 95% buffer B and reequili-brated with buffer A. Mass spectra were acquired in a data-dependent manner, with an automatic switch between MS and MS/MS scans. High-resolution MS scans were acquired in the Orbitrap(60,000 FWHM, target value 106) to monitor peptide ions in themass range of 350–1650 m/z, followed by CID MS/MS scans in theion trap (minimum signal threshold, 150; target value, 10,000; iso-lation width, 2m/z) of the five most intense precursor ions. To avoidmultiple scans of dominant ions, the precursor ion masses ofscanned ions were dynamically excluded from MS/MS analysis for15 s. Singly charged ions and ions with unassigned charge stateswere excluded from MS/MS fragmentation.Raw data were converted to the open mzXML format with

ReAdW (version 4.3.1). mzXML files were searched by theSEQUEST search engine against UniProtKB/Swiss-Prot proteindatabase version 57.15 of M. musculus concatenated with the se-quences of common contaminants. Search parameters for the pep-tide identification included a precursor mass tolerance of 0.05 Da, aminimum of one tryptic terminus, and a maximum of two internaltrypsin cleavage sites. Cysteine carbamidomethylation (+57.021 Da)was set as a static amino acid modification, and methionine oxida-tion (+15.995 Da) and asparagine deamidation (+0.9840 Da) wereset as differential modifications. The PeptideProphet and Protein-Prophet tools of the TPP (version 4.5) were used for probabilityscoring of peptides and proteins, and identifications were filtered toan FDR of ≤1%. For the analysis of NP41-captured glycopeptides,peptide features were filtered for the presence of the N[115]-X-S/Tmotif introduced by PNGaseF cleavage (wherein N[115] stands for adeamidated asparagine residue, X for any amino acid except proline,and S/T for serine or threonine). Peptides and proteins werequantified by label-free quantification using the Progenesis (Non-linear Dynamics) software package.

Immunofluorescence.Healthy or degenerated facial nerves (6 wk or2.5 mo posttransection) in surrounding muscle were fixed in 4%paraformaldehyde in PBS, blocked with 5% normal goat serum(Invitrogen), and stained with the primary antibodies listed inMaterials and Methods. Signal was detected with AlexaFluor488anti-rabbit and -rat antibodies (Invitrogen) and imaged withNikon confocal fluorescence microscope. Serial sections stainedwith H&E were imaged with white light microscopy.

Phage-Binding ELISA. The 96-well plates were coated with purifiedrecombinant mouse laminin-α4 subunit, human nidogen-1, mousenidogen-2, or no protein as a control at 10 μg/mL in 2 M sodiumbicarbonate, pH 9.5. ELISA was performed as described inMaterials and Methods. Multiple measurements were taken every5 min over the course of HRP development, each normalized bybackground subtraction and expressed as a percentage relative tothe maximum signal. All measurements except at time 0 wereaveraged.

Peptide Synthesis and Labeling. All NP41 peptides (acetyl-SHSNT-QTLAKAPEHTGC-amide) were labeled on the C terminus cys-teine by maleimide derivatives of MB (AttoTec), DBF, eosin (LifeTechnologies), or biotin (Sigma-Aldrich). NP41–FAM was pre-pared as previously described. Peptides were purified to >95%purity using C-18 reverse-phase HPLCy (HPLC) with a 20–50%acetonitrile gradient in 0.05% TFA and confirmed by MS [allconcentration percentages expressed as (vol/vol)].5,6-carboxy-4′,5′-DBF. To a stirred suspension of 5,6-FAM (376 mg,1 mmol) in 80% aq. HOAc (5 mL) at room temperature, bromine(102 mL, 2 mmol) was added dropwise. The solid dissolved, andafter stirring overnight, the crude product was collected by fil-tration, washed with water, and dried in vacuo over P2O5 (Yield,0.5 g). LC–MS revealed the desired product (56% by absorbance

Glasgow et al. www.pnas.org/cgi/content/short/1611642113 2 of 8

at 254 nm) with unreacted starting material (1%) and mono-(5.5%), tri- (15.6%), and tetrabrominated (21.7%) products andwas used without further purification [ES-MS(M+H+), 534.8;calculated, 534.9].5,6-carboxy-4′,5′-DBF N-hydroxysuccinimide ester (DBF-NHS). Crude 5,6-carboxy-4’,5′-DBF (200 mg, 0.37 mmol) was dissolved in anhy-drous THF (10 mL) under N2, and NHS (52 mg, 0.45 mmol) wasadded followed by diisopropylcarbodiimide (DIPC; 59 μL,0.38 mmol). After 4 h of stirring at room temperature, LC–MSindicated an incomplete reaction, so an additional 25 μL ofDIPC was added. After overnight incubation, the reaction mixwas evaporated, dissolved in dry DMSO (1 mL), filtered, andpurified by prep HPLC eluting with a gradient of 10–90% ace-tonitrile–water–0.05% TFA for 20 min. The collected fractionswere pooled and immediately frozen and lyophilized to give ared solid (ES–MS(M+H+), 631.9; calculated, 632.9).L-NP41–DBF–biotin–pyridylhydrazine. NH2–SHSNTQTLAKAPEHT-GCE(biotinyl-PEG)–CONH2 was synthesized by standard Fmocchemistry on NovaSyn TGR resin using Fmoc-glu(biotinyl-PEG)–OH (Novabiochem). The washed and dried resin-boundpeptide (160 mg, 25 μmol nominal) was suspended in dry DMF(1 mL), and DBF–NHS ester (15 mg, 24 μmol) was added with4-methylmorpholine (50 μL, 0.45 mmol). After stirring overnightat room temperature, the product was collected by filtration,washed with DMF (3 × 5 mL) and methanol (3 × 5 mL), anddried in vacuo. Following cleavage for 2 h with 2 mL of freshlyprepared cleavage mixture (2.5% water, 2.5% thioanisole, 2.5%TIPS, 2.5% EDT, 90% TFA) and removal of the resin by fil-tration, the peptide was precipitated with cold diethyl ether-

hexane (1:1 vol/vol) and collected by centrifugation. The productwas purified by prep reverse-phase HPLC using a gradient ofacetonitrile–water with 0.05% TFA to yield 11.1 mg L- and11.8 mg D-peptides [ES–MS(M+3H+), 952.6; calculated, 952.6;(M+4H+), 714.8; calculated, 714.7].The peptide was dissolved in 50% ACN–water–0.1% TFA

(0.5 mL, ∼8 mM) and treated with maleimide–peg2–pyridylhydrazineacetone hydrazone (70 μL, 100 mM solution in DMSO) andK.Mops (100 μL, 0.1 M in water pH 7.2). After 1 h at room tem-perature, acetic acid (100 μL) and acetone (50 μL) were addedand kept overnight at 4 °C. The product was purified by prepHPLC to yield 11.5 mg [ES–MS(M+3H+), 1,110.7; calculated,1110.4; (M+4H+), 833.4; calculated, 833.1].D-NP41–FAM–biotin–pyridylhydrazine. NH2–shsntqtlakapehtgcE(bio-tinyl-PEG)–CONH2 was synthesized using the procedure forL-NP41–DBF–biotin–pyridylhydrazine [ES–MS(M+3H+), 1,110.9;calculated, 1,110.4; (M+4H+), 833.4; calculated, 833.1].NP41–FAM–biotin–pyridylhydrazine. NH2–SHSNTQTLAKAPEHT-GK(FAM)CE(biotinyl-PEG)–CONH2 was synthesized by stan-dard Fmoc chemistry on NovaSyn TGR resin using Fmoc-glu(biotinyl-PEG)–OH (Novabiochem) and Fmoc-lys(5,6-FAM)(Anaspec). The washed and dried resin-bound peptide (25 μmolnominal) was suspended in dry DMF (2 mL), and N-acetoxy-succinimde (100 mg, 0.64 mmol) was added with 4-methyl-morpholine (100 μL, 0.91 mmol). The procedure from L-NP41–DBF–Biotin–pyridylhydrazine was then followed [ES–MS(M+3H+),1,114.7; calculated, 1,114.7; (M+4H+), 836.6; calculated, 836.3].Partial loss of acetone occurs to the free hydrazine [ES–MS(M+3H+),1,101.2; calculated, 1,101.3; (M+4H+), 826.4; calculated, 826.3].

Glasgow et al. www.pnas.org/cgi/content/short/1611642113 3 of 8

@ = +4 Da ^ = +16 ~ = +32

Modified tryptophans identified by SEQUEST:

A

0.00

0.50

1.00

1.50

2.00

2.50

3.00

30 0 3 10 30 90

YDSA... INTQ... NAHS...

Seconds of illumination

perc

enta

ge

B

No MB-biotin control

Kynurenine (+4)

0.00

1.00

2.00

3.00

4.00

5.00

30 0 3 10 30 90

YDSA... INTQ... NAHS...

Seconds of illumination

perc

enta

ge

C

No MB-biotin control

Tryptophan-OH (+16)

Streptavidin sequence

Fig. S1. Photooxidation of MB–biotin results in tryptophan oxidation of streptavidin. (A) Primary sequence of streptavidin with observed tryptic peptidesunderlined; tryptophans are highlighted and modified listed with mass identified by the SEQUEST algorithm. (B) Percent of peptides containing +4 kDatryptophan mass change (kynurenine) increases with photooxidation. The area of the modified peak is plotted as a percentage of area of unmodified peak.Peak conversion occurred after 30 s of light exposure. (C) Percent of peptides containing +16 kDa tryptophan mass change increases with photooxidation. Peakconversion occurred after 3 s of light exposure.

Glasgow et al. www.pnas.org/cgi/content/short/1611642113 4 of 8

0

300

600

0.5 0.75 1

Tota

l spe

ctra

l cou

nts

(PO

+NL)

15 min photooxidation

0

250

500

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Tota

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(PO

+NL)

1 min photooxidation

0

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500

0.5 0.75 1

Tota

l spe

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(PO

+NL)

10 s photooxidation

Col6a3 Col6a1 Col6a2

Lamc1 Lama2

Nid1

Lamb1 Lamb2

Lama4

Lamc1 Nid1 Lamb2

Lama4

Col6a3

Col6a1

Col6a2 Lamc1

Lama2

Nid1 Lama4 Lamb1

Col6a1

Col6a2

Col6a3

Enrichment PO/(PO+NL) Enrichment PO/(PO+NL) Enrichment PO/(PO+NL)

Lamb2

Spectral Counts 10 s 1 min 15 min

X Y X Y X Y

Lamb2 0.79 67 0.71 48 0.77 60 Lama4 0.72 36 0.68 31 0.74 39 Nid-1 0.66 92 0.62 81 0.64 85 Lamb1 0.65 31 0.54 24 0.73 40 Lama2 0.63 110 0.43 72 0.59 99 Lamc1 0.57 104 0.54 98 0.6 112 Col6a3 0.54 424 0.51 398 0.6 496 Col6a1 0.54 140 0.54 140 0.63 174 Col6a2 0.53 103 0.51 98 0.62 125

100

200

300

400

500

0.4 0.6 0.8

Tota

l spe

ctra

l cou

nts

(PO

+NL)

Avg. photooxidation enrichment Avg PO/(PO+NL)

Col6a3

Col6a1 Col6a2 Lamc1

Lama2 Nid1

Lamb2 Lamb1 Lama4

Nfml

Mpz

Average (10 s, 1 min, 15 min photooxidation vs. no light)

Fig. S2. Ligand proximal photoxidation of NP41–MB enriches laminins after various photooxidation exposures. Shown are proteins identified as enriched byMS in nerve samples photooxidized with NP41–MB after light exposure (10 s, 1 min, or 15 min) versus NL control, plotted by average (Top) or individual lightexposures (Middle). Spectral counts for each protein were summed [abundance (PO+NL)], and the fractions identified in the PO sample [enrichment PO/(PO+NL)] are plotted on the y and x axes, respectively. Proteins enriched in the PO compared with the NL control are shown on the right of the vertical axis at x =0.5. Proteins with low total spectral counts (<30) were considered likely nonspecific background signal. Those identified as consistently enriched by light (x >0.5) and with abundant spectral counts (y > 30) are highlighted in the graphs above and are identified with x and y values listed in the table. Proteins showingphotooxidation-induced enrichment in multiple samples include laminin subunits, nidogen, and collagen VI subunits. Two abundant, nerve-specific proteins,neurofilament and myelin protein P0, were not enriched. Results are representative of independent replicate experiments.

Glasgow et al. www.pnas.org/cgi/content/short/1611642113 5 of 8

NP41-MB

Biotin-NP41

Structure shown in Fig. 3

NP41-DBF

NP41-FAM

Ex vivo staining shown in Fig. 2 and Fig. 5

In vivo staining shown in Fig. 6a

Not fluorescent

Fig. S3. NP41 conjugated to various SOGs and biotin shows consistent perineurial localization, whereas in vivo nerve-to-muscle contrast differs betweenconjugates. (Left) NP41 conjugates and structures. (Middle) NP41–MB and NP41–DBF treated on nerve sections and imaged by fluorescence show perineurialstaining (highlighted areas). NP41–biotin treated on tissue sections and stained by streptavidin–HRP and DAB shows strong perineurial signal (darkened areas).(Scale bar, 500 mm.) Gain levels are set individually. (Scale bar, 50 mm.) (Right) NP41 dyes, injected systemically into mice and imaged 2–4 h after, showdifferent sciatic nerve contrast in vivo. NP41–MB showed low nerve fluorescence in vivo that was difficult to distinguish from the surrounding muscle. NP41–DBF showed higher nerve fluorescence and contrast to muscle (n = 2).

Glasgow et al. www.pnas.org/cgi/content/short/1611642113 6 of 8

NP41 dibromofluorescein pyridylhydrazineA

0

160

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0.00 0.50 1.00

Tota

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Photooxidation enrichment

PO/(PO+NL)

Control peptide: D-NP41-DBF-HB

Col6a3

Nid1

Lamc1 Lama2

Col6a1

Col6a2 Lama4

Hspg2

Lamb2

Nfml

Mpz

B

Nid2 40

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Photooxidation enrichment PO/(PO+NL)

NP41-DBF-HB

Col6a3

Nid1 Lama2 Lamc1 Lamb2

Col6a1 Col6a2

Lama4 Lama5

Nfml

HspgMpz

Lamb1

NP41-DBF HB photooxidizedvs. D-NP41 photooxidized control

Protein

Enrichment NP41PO/ (NP41PO+D-

NP41PO)

Abundance NP41PO+D-

NP41PO

laminin subunit alpha-5 1 1 nidogen-2 0.83 23 laminin subunit beta-1 0.83 18 nidogen-1 0.79 103 laminin subunit alpha-2 0.78 64 laminin subunit alpha-4 0.76 17 laminin subunit beta-2 0.73 48 laminin subunit gamma-1 0.69 62 basement membrane-specific heparan sulfate proteoglycan core protein 0.67 27 collagen alpha-3(VI) 0.62 198 collagen alpha-1(VI) 0.57 35 collagen alpha-2(VI) 0.52 27

D

NP41-DBF-HB in vivo NP41-MB ex vivo

PO vs. NL NP41PO vs. D-NP41PO

control L: PO vs. NL

PO/(NL+PO) NL+PO

NP41PO/ (NP41PO

+D-NP41PO)

NP41PO+D-

NP41PO

PO/(NL+PO) NL+PO

lumican precursor 0.93 15 0.7 20 0 3

collagen alpha-2(I) 0.82 34 0.62 45 0.73 15

testis-specific serine/threonine-protein kinase 4 isoform X1 0.76 75 0.83 69 0 0

vimentin 0.72 64 0.66 70 0.56 129

collagen alpha-1(I) 0.69 97 0.66 101 0.54 22

E C

NP41-DBF-HB Control peptide D-NP41-DBF-HB

Protein Enrichment

PO/ (PO+NL)

Abundance NL+PO

Enrichment PO/

(PO+NL)

Abundance NL+PO

nidogen-2 1 19 1 4 laminin subunit alpha-4 1 13 0.8 5 laminin subunit alpha-5 1 1 0 laminin subunit gamma-1 0.88 49 0.76 25

laminin subunit beta-1 0.88 17 1 3

laminin subunit alpha-2 0.83 60 0.61 23 nidogen-1 0.8 101 0.61 36 laminin subunit beta-2 0.78 45 0.62 21 basement membrane-specific heparan sulfate proteoglycan core proteiin

0.78 23 0.64 14

collagen alpha-1(VI) 0.67 30 0.71 21 collagen alpha-3(VI) 0.61 200 0.6 126 collagen alpha-2(VI) 0.61 23 0.87 15 neurofilament light polypeptide 0.51 316 0.358 176

myelin protein P0 0.44 50 0.722 18

00000000000

160

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+NL)

TT

Photooxidation enrichment

PO/(PO+NL)

Control peptide: D-NP41-DBF-HB

Col6a3

Nid1

LamLama

CoLa

Nfml

Mpz

Nid2 40 44

80

120

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200

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280

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.00 0.50 1.00

Tota

l spe

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+NL)

TT

Photooxidation enrichment PO/(PO+NL)

NP41-DBF-HB

Col6a3

Nid1Lama2 Lamc1Lamb2

Col6a1Col6a2C

Lama4 Lama5

Nfml

HspgMpz

Lamb1

0

80

120

40

Fig. S4. Ligand proximal photooxidation in vivo with NP41–DBF pyridylhydrazine–biotin (NP41–DBF–HB) conjugate shows enrichment of laminin, collagen,and associated proteins. (A) Structure of NP41–DBF–HB conjugate. (B) Relative quantification of spectral counts in PO sample versus NL control for each proteinidentified by MS. (Left) In vivo photooxidation of DBF–NP41–HB shows high enrichment of basement membrane proteins, including laminin subunits andnidogens. Collagen VI subunits showed high abundance and low enrichment, and laminin a5 showed high enrichment but low abundance. Two abundant,nerve-specific proteins, neurofilament and myelin protein P0, were not enriched. (Right) By comparison, in vivo photooxidation of a control conjugate with allD-amino acids showed low enrichment and low abundance of the same proteins. (C) Table of values of proteins identified in the graph, showing x and y valuesof each identified protein. (D) Relative spectral counts of a photooxidized NP41–DBF–HB conjugate compared with a photooxidized d-NP41 control conjugate,

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showing enrichment and total abundance of identified proteins. Laminin subunits and nidogen showed the highest target-specific enrichment with abundantspectral counts. Laminin-α5 showed high enrichment but low spectral counts, and collagen subunits showed high spectral counts but low enrichment.(E) Relative quantification of other proteins slightly enriched in the NP41–DBF–HB experiment not identified in NP41–MB as enriched and abundant. Proteinsshowing moderate enrichment (>0.69) upon photooxidaton of NP41–DBF–HB compared with NL control had low enrichment in comparison with the pho-tooxidized D-amino acid control, except for testis-specific ser/thr kinase and lumican. In the MB–NP41 photooxidation experiment, the former was notidentified and the latter was unenriched.

NP41-FAM-HB

100

mM

N

aIO

4 w

ater

White light Fluorescence

B

NP41-FAM

A NP41-FAM-HB

Fig. S5. NP41–FAM–HB localizes to perineurium and cross-links to the nerve after treatment with periodate. (A) Nerves from NP41–FAM–HB- or NP41–FAM-treated mice show similar fluorescence localization with high perineurial staining. (B) Fluorescence is retained in nerves from animals injected with NP41–FAM–

HB after periodate glycan oxidation and extensive washout but not in control nonperiodate-treated nerves, indicating glycan-mediated cross-linking.

0%

20%

40%

60%

80%

100%

Lama4 subunit

Nid1 protein Nid2 protein no protein

Aver

age

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enta

ge o

f max

imum

NP41 X12 library

*

Fig. S6. NP41 binds to purified laminin-α4 subunit. ELISA of NP41-expressing phage shows significantly greater binding to purified laminin-α4 subunit (re-combinant mouse isoform) than phage expressing a library of random 12-amino acid peptides (*P < 0.01). Binding to nidogen-1 (Nid1, human recombinantisoform) and nidogen 2 (Nid2, mouse recombinant isoform) was not significantly greater than the control. The mean signal, background-subtracted from noprotein control, is graphed as a percentage of the maximum signal in two independent experiments, each performed in triplicate. Error bars represent SD.Paired two-tailed t test, P = 0.007 Lama4, P = 0.13 Nid1, and P = 0.16 Nid 2.

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