Evaluation and improvement of protein extraction methods for analysis of skin proteome by noninvasive tape stripping

Journal Pre-proof Evaluation and improvement of protein extraction methods for analysis of skin proteome by noninvasive tape stripping

Patrick Kaleja, Hila Emmert, Ulrich Gerstel, Stefan Weidinger, Andreas Tholey PII:

S1874-3919(20)30046-4

DOI:

https://doi.org/10.1016/j.jprot.2020.103678

Reference:

JPROT 103678

To appear in:

Journal of Proteomics

Received date:

20 November 2019

Revised date:

10 January 2020

Accepted date:

2 February 2020

Please cite this article as: P. Kaleja, H. Emmert, U. Gerstel, et al., Evaluation and improvement of protein extraction methods for analysis of skin proteome by noninvasive tape stripping, Journal of Proteomics (2020), https://doi.org/10.1016/j.jprot.2020.103678

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2020 Published by Elsevier.

Journal Pre-proof

Evaluation and improvement of protein extraction methods for analysis of skin proteome by noninvasive tape stripping

Patrick Kaleja1 , Hila Emmert2 , Ulrich Gerstel2 , Stefan Weidinger2 , and Andreas Tholey1 *

Institute for Experimental Medicine,

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Systematic Proteome Research & Bioanalytics,

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1

Department of Dermatology and Allergy, University Hospital Schleswig-Holstein, Campus

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2

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Christian-Albrechts-Universität zu Kiel, Kiel, Germany

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Kiel, Germany

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* to whom correspondence should be addressed:

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Andreas Tholey

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Systematic Proteome Research & Bioanalytics, Institute for Experimental Medicine Christian-Albrechts-Universität zu Kiel 24105 Kiel, Germany

Phone: #49 (431) 50030300; Fax: #49 (431) 50030308 E-mail: [email protected]

Journal Pre-proof Abbreviations

TS

Tape strip

SP3

Single-pot, solid-phase-enhanced sample preparation

Keywords

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Clinical proteomics, biopsy, biomarker, skin, dermatitis

Journal Pre-proof Abstract Analysis of the human skin proteome is key to understand molecular mechanisms maintaining health or leading to diseases of this important organ. For minimal invasive sampling of skin proteomes, the use of self-adhesive tape strips has been successfully applied. However, the methods previously presented were evaluated on different types of skin samples (e.g. healthy, diseased) and used a variety of cell lysis/protein extraction methods, which renders a systematic comparison and thus the identification of the most

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efficient protocols difficult. Here, we present a study comparing five different approaches for cell lysis and protein

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extraction from single tape strip biopsies. Extraction using a detergent mix or 1% SDS proved to be most efficient. Further, we replaced protein precipitation by single-pot, solid-

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phase-enhanced sample preparation (SP3), which strongly enhanced the number of

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identified proteins. This fully LC-MS compatible methodology provides a fast and

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Biological Significance:

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reproducible approach for minimal invasive sampling of human skin proteomes.

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Fast and reproducible minimal invasive sampling of human skin proteomes is a major prerequisite for clinical proteomics studies aiming to decipher molecular mechanisms involved in the homeostasis as well as in the development of diseases. By optimization of tape strip sampling, e.g. the introduction of SP3 sample cleanup prior to LC-MS analysis, the presented protocol leads to yet not reported numbers of protein identifications from healthy human skin. Further, due to its efficiency it allows analysis from minimal sample amounts, e.g. from single tape strips, while established protocols relied on pooling of multiple tape strips. This provides the opportunity to perform spatially (lateral) resolved proteome analyses from different depths of the skin by analysis of consecutive strips.

Journal Pre-proof Introduction The human skin is the outer barrier organ, which protects the organism from external factors by a number of mechanisms, in particular via physicochemical and immunological processes. To better understand homeostatic, as well as disease related molecular mechanisms, knowledge of the proteome composition of the skin under different biological situations is key. For the outer layers of the skin (stratum corneum), sampling using self-adhesive tape strips

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(TS) has become a major method for analytical purposes. This method is easy to perform and minimal invasive and therefore relatively smooth for patients.[1]

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TS are small polymer based adhesive strips, which are available in different sizes. In most

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cases, tape strips are repetitively applied on the same spot on a patient’s skin, in order to sequentially remove layers of extracellular matrix and embedded cells, which remain on the

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tape.[2] TS were used for analysis in different studies, e.g. in pharmacological and

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toxicological reagents penetration assays,[3] analytics of lipids[4] and increasingly for the analysis of skin proteins, e.g. for LC-MS based studies. For instance, Azimi et al.

spectrometry.[6]

stratum

corneum

via TS, including protein based mass

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al. analyzed psoriatic

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characterized patients with actinic keratosis, which are premalignant tumors,[5] and Méhul et

Protein amounts extracted from TS differ depending on multiple parameters such as individual skin characteristics and the localization of the acquired area. However, it is generally estimated that the amount of one TS is in the range of 1 mg/cm 2 stratum corneum,[7] while the protein concentration, mostly estimated via Keratin, is in the range of multiple 100 µg.[3] Harsh conditions with high salt and/or detergent concentrations are required for extraction of proteins and other molecules from TS samples, as non-covalent interactions to adhesive have to be overcome and highly cornified cells have to be lysed. In order to remove these

Journal Pre-proof additives, protein precipitation is often used, which can however result in significant sample loss. Hence, analysis of intact skin samples yields in many cases relatively low numbers of protein identifications. For example, Ma et al. reported 151 identified proteins from intact patient derived samples.[8] In diseased samples, higher numbers have been identified, however, here also proteins deriving from tissue repair processes or open wounds (e.g. blood proteins) were identified; for example, in samples of actinic keratosis, around 630 proteins could be identified.[5] In order to improve the number of identifications, many

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protocols either rely on very long extraction times (more than 24 h for single tape strips),[8]

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or on the combination of multiple TS or strips with large surface areas to increase the

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amount of starting material.[9,10] Therefore, with only few exceptions,[11] tape stripping was

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only rarely used as a low sample amount analytical strategy, e.g. analysis of single TS. We here report a study for the first time comparing different approaches of cell lysis/protein

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extraction from TS by means of different buffers and detergents on slices of identical tape strips. For sample cleanup, we introduce the “single-pot, solid-phase-enhanced sample

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preparation” (SP3) approach.[12,13] A major emphasis was laid on the use of single TS (or

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only parts of it) to reduce sample amounts and the efforts for sampling of patient materials.

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The study led to the identification of suitable protocols compatible with subsequent LC-MS analysis of the skin samples.

Materials and Methods Chemicals Trypsin/LysC Mix (Mass Spec Grade) was purchased from Promega (Madison, WI, USA) and D-squame Tape Strips (TS) purchased from Clinical and Derm (Dallas, Texas, USA). The term tape strip in this publication relates to this type of commercial product. Deionized water (18.2 MΩ/cm) was prepared by an arium 611VF system (Sartorius, Göttingen, Germany). SP3 beads were purchased as Sera-Mag SpeedBead Carboxylate-Modified

Journal Pre-proof Magnetic Particles by GE Life Sciences (Little Chalfont, UK). Other chemicals were purchased from Sigma Aldrich (Munich, Germany).

Tape stripping procedure Tape strip samples were collected from healthy volunteers (n = 5; 3 females, 2 males; age: 23-32; method development part: 1 male) with no history of chronic skin disease under an Institutional Review Board-approved protocol (A110/12). Volunteers were advised not to use

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skin creams or similar products 24 h prior to sample collecting. Tape strips were applied on

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forearm of each volunteer in consecutive order at an identical spot. Strips were applied for

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10 s with a constant pressure of 225 g/cm 2applied by a pressure instrument (Clinical and Derm, Texas, USA), with the first strip being discarded to avoid any contamination. Samples

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were subsequently transferred in 1.5 mL reaction tubes (Eppendorf, Germany) and stored at

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-80 °C till usage. As a negative control, blank tape strips were used.

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Protein extraction from tape strip samples

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For comparison of different protein extraction methods, single TS (having 2.2 cm diameter and 3.8 cm 2 surface area) were cut into five equal pieces using PTFE coated scissors and

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their relative mass measured via gravimetrical approach to adjust the following sample injection volumes. Samples were handled in technical triplicates by using consecutive strips and each piece transferred into 500 µL reaction tube and 100 µL corresponding lysis buffer added. Each buffer/extraction solution consisted of 100 mM HEPES pH 7.5 (adjusted with NaOH) with 1x cOmplete Mini EDTA-free protease inhibitor cocktail and: method A: 0.1% RapiGest; method B: 4% SDS; method C: 1% SDS, 1% Triton X100, 1% NP-40, 1% Tween 20, 1% Deoxycholate, 50 mM NaCl, 5 mM EDTA; method D: water; method E: 6M guanidinium hydrochlorid (GndHCl) (Table 1). To test optimal SDS concentrations for protein extraction, samples were handled identical except the adjustment of final SDS concentration to 4%, 2% and 1% in triplicates. For comparative analysis of volunteer samples, consecutive TS were used from each person

Journal Pre-proof and sliced into half. Pieces were transferred into reaction tubes and 100 µL of extraction buffer (method B or C) added.

Samples were heated to 96°C for 10min, except for mix samples (method C) which were heated to 56°C for 30min; the reduced temperature was chosen as Tween20 can be degraded at elevated temperatures according to manufacturer’s product sheet. Samples were cooled to 20 °C and sonicated for 10min. Proteins were reduced with 12 mM tris(2-

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carboxyethyl)phosphine (TCEP) and 40 mM chloroacetamide (CAA) at 25 C for 1h.

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Following, proteins were enriched and desalted via SP3-protocol.[13] SP3-beads were

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prepared by mixing 100 µL of hydrophilic with 100 µL of hydrophobic coated beads in solution, washing two times with ddH2O and resuspension in 500 µL ddH2O (final

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concentration: 20 µg/µL). Per sample, 10 µL of beads were added and resuspended by

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gentle pipetting. To induce protein binding, ethanol was added to final concentration of 50% (v/v). Samples were mixed at 25°C for 5min to ensure complete binding, beads immobilized

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via magnetic rack and the supernatants removed. Beads were washed two times by adding

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180 µL 80% ethanol and finally the supernatant was completely removed. Proteolytic digestion was induced by resuspending the beads in 100 mM ammonium bicarbonate buffer

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pH 8 with Trypsin/LysC Mix (enzyme to protein ratio of 1:50 (wt/wt); total protein amounts extracted from full tape strips were determined in a pre-experiment using the BCA-assay); according to the value determined for the method C, the ratio was set to 1:50; the same amount of protease was added for all other extraction methods, which leads to increased enzyme to protein ratios, e.g. for methods D and E. Beads were shortly sonicated to ensure resuspension and digestion performed at 37°C and 800 rpm mixing. Digested peptides were desalted with C18 pipette tips (Pierce C18 tips 10 µL, Thermo Scientific) according to manual and redissolved in 3% ACN, 0.1% TFA for downstream mass spectrometry analysis.

Journal Pre-proof For a pre-experiment using RapiGest, buffer (method A), consecutive TS were sliced in five pieces and each replicate lysed, as mentioned before, in RapiGest containing buffer. Samples were reduced/alkylated, digested and samples either (i) cleaned up via SP3 method (see above) or (ii) RapiGest precipitated via acidifying solutions to pH 2 using TFA and incubation at 37°C. Precipitated RapiGest was removed by centrifugation and supernatant desalted using C 18 pipette tips.

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For comparison of SP3 protocol to protocols including protein precipitation, a single tape

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strip was used with one blank tape strip as negative control. Both TS were sliced into smaller

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pieces, and subsequently lysed and reduced/alkylated as described before in 300 µL of extraction buffer (method B). Samples were split into five equal aliquots, with three used for

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protein precipitation prior to digestion. Precipitation was performed by adding 9 volumes of

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cooled ethanol, vigorous vortexing and incubation at -80 °C for 16 h. Precipitates were centrifuged (21.1 x 103 g at 4°C for 20 min), washed two times with ethanol and dried.

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Protein pellets were resuspended for digestion by pH shift using 50 mM NaOH, with

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subsequent dilution in 100 mM ammonium bicarbonate. Samples were then digested and

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desalted as described above.

Liquid chromatography-mass spectrometry Resolubilized peptides were analyzed by reverse phase low pH nanoHPLC online coupled to an

Orbitrap

Q Exactive

Plus

Mass

Spectrometer

(Thermo

Scientific).

Prior

to

chromatographic separation, peptides were desalted on a C18 trapping µ-precolumn Acclaim PepMap 100 with 300 µm i.d. x 5 mm, 100 Å pore size and 5 µm particle size (Thermo Scientific). Nano-LC was performed on an Acclaim PepMap RSLC 75 µm x 50 cm nanoViper column (Thermo Scientific) with a Dionex Ultimate 3000 System (Thermo Scientific) and eluent A (0.05% formic acid in deionized water) and B (80% acetonitrile, 0.05% formic acid in deionized water). Gradient started at 5% eluent B up to 2min, following a linear increase to 40% at 62min, 95% at 67min and keeping this isocratic for 10min; the flow rate was 300

Journal Pre-proof nL/min. For online measurements, a data dependent acquisition method with top 10 precursors for higher energy collisional dissociation (HCD) fragmentation at 29% normalized collision energy in positive ion mode was utilized. Peptide fragmentation of charge states +2 to +8 was enabled, and resolution on MS1 was set to 70,000 with a scan range from 300 to 1,800 m/z and on MS2 to 17,500. AGC target was set to +3 e6 on MS1 level and +1 e5 on MS2, respectively. Dynamic exclusion list was used with 40 s cycles and exclusion of isotopes set true. The isolation window was selected with ±1.5 m/z.

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Data analysis was performed on Proteome Discoverer 2.2 (Thermo Scientific) with

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SequestHT search nodes. Variable modifications were set with oxidation on methionine

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[+15.9949 Da], acetylation on N-termini [+42.0106 Da] and deamidation/citrullination on asparagine, glutamine and arginine [+0.9840 Da], while carbamidomethylation on cysteine

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[+57.0215 Da] was set as fix modification. Enzyme specificity was set to semi-tryptic with

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maximum two missed cleavages. Precursor mass tolerance was set to 10 ppm and fragment mass tolerance to 0.02 Da. Data were searched against a database consisting of 20,386 proteins

and

additional

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common

contaminating

proteins

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human

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(https://www.uniprot.org/uniprot/; only reviewed sequences, 31st of July 2018). For FDR validation, Percolator node with target FDR of 0.01 based on q values was set as a high

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confidence filter. Protein identifications of volunteer experiments were further evaluated using String network analysis, including GO term annotation.[14] All LC-MS data have been deposited to the ProteomeXchange Consortium[15] via the PRIDE partner repository with the dataset identifier PXD016319. Instrument data were converted to prot.xml and pep.xml via Proteome Discoverer 2.2.

Results and Discussion Evaluation of different TS extraction protocols and introduction of SP3 sample cleanup

Journal Pre-proof Self-adhesive TS provide an efficient opportunity to obtain protein samples from skin. After sampling process, two major steps are key for a successful application of this approach. First of all, cells adhered to the TS have to be efficiently lysed and proteins extracted. For this

purpose, different buffers, chaotropes and detergents/surfactants have been

successfully applied to break up cells and to solubilize the proteins. Second, in order to be compatible with downstream LC-MS analysis, together with cell debris and nonproteinogenic compounds (lipids, others), the detergents, buffers and chaotropes have to be

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removed. Removal of detergents/salts by precipitation (e.g. acetone or methanol/chloroform)

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particular when applied to small sample amounts.

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has been applied successfully.[16] However, this process is known to lead to sample loss, in

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An alternative is the use of cleavable detergents, e.g. RapiGest, which is acid cleavable and was previously used on TS.[5] After acidification, the cleavage products are removed by

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centrifugation; however, this process has been shown to be critical when very small sample volumes have to be treated.[17] We therefore splitted slices of skin sample loaded TS into

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two groups and extracted the proteins using 0.1% (w/v) RapiGest. Samples were then either

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(i) digested in solution with subsequent removal of RapiGest by acidification according to the

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standard protocol or (ii) cleaned using the established SP3 sample protocol using magnetic beads;[13] this step also includes the digestion of the proteins directly from the beads . LCMS results of these two approaches are shown in Fig. 1A and B (Suppl. Table 1 & 2). We observed a clear increase in protein identifications from 86±34 protein groups by the standard acid cleavage/centrifugation protocol compared to over 264±32 protein groups with SP3 protocol (408±204 compared to 1921±586 peptide groups; average values with standard deviation). Venn diagrams of protein identifications between both conditions show further the acidic precipitation protocol as a subset of the SP3 sample cleanup, which proves it to be the superior strategy (Fig 1B). Interestingly, SP3 sample cleanup also showed an increase in protein IDs in control samples (blank TS), which presumably better reflects the identifiable proteins on blank TS. These

Journal Pre-proof consisted mostly of Keratins, but also other structural proteins such as Desmoplakin (Suppl. Table 1).

As the SP3-supported sample cleanup proved to be successful in combination with the RapiGest protein extraction (method A), we tested its combination with another two recently established and two novel extraction approaches (Table 1): method B: use of 4% (w/v) SDS; the use of this detergent was reported in multiple strategies with concentrations varying from

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1-4%; method C: use of a multi detergent mix, composed of 1% SDS, 1% Triton X100, 1% NP-40, 1% Tween 20, 1% Deoxycholate, 50 mM NaCl and 5 mM EDTA; this condition

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mimics approaches using different detergents mixes such as SDS/butylated hydroxytoluene

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or the use of other commonly used detergents as Triton [16,18]: the detergent mix applied here was described to be fully compatible with SP3;[13] method D: use of a buffer solution

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composed by Clausen et al.;[19] method E: the use of a buffered guanidine hydrochloride

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solution; the exclusive usage of chaotropes for tape strip extraction has not been shown yet.

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In order to allow a direct comparison of these five approaches, collected skin samples on TS were divided in five different slices of equal sizes using PTFE coated scissors; the weight of

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the TS slices was used as a control for equal amounts. The five extraction methods were tested in three technical replicates, using three consecutive TS-biopsies to avoid significant differences in protein composition in different layers of the skin. The number of peptides and protein groups identified by LC-MS was used as readout of the success of the extraction process (Suppl. Table 3 & 4).

As shown in Fig. 2A and B, extraction strategies using detergent-free buffers or chaotropes (methods D and E, respectively) were less efficient to extract proteins from TS compared to those employing detergents. In an pre-experiment, we determined the protein concentrations extracted with the five methods by using the BCA assay. In order to reduce the amount of

Journal Pre-proof detergents and chaotropes interfering with the BCA assay, the samples were precipitated with ethanol prior to the assay. For methods D and E, we determined protein concentrations below 50 µg/mL, whereas the detergent based methods A-C delivered 5-10 times higher concentrations. Note that these values are not accurate protein concentrations but are sufficient to reflect the efficiency of the extraction procedures. Protein identification numbers of methods D and E, respectively, were significantly below those achieved in detergent-based extraction buffers (494±170 and 516±67 peptides and

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122±36 and 82±4 protein groups in methods D and E, respectively). Proteins identified in buffer/chaotrope treated samples were mostly also identified in the detergent treated

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samples; only 12 proteins were identified solely in buffer, and only a single protein

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exclusively in the chaotrope containing buffer or the buffer and chaotrope condition. We did not adapt the protein amounts injected to the LC-MS system to balance the

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differences determined by BCA assays described above. This is justified as we wanted to

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compare the efficiency of overall workflow, finally expressed by the number of protein identifications achievable with the different extraction methods. Thus, different protein

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amounts were loaded on the SP3 beads. After subsequent digestion and C18-desalting,

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equal volumes of the digest were injected. Overall, methods B (4% SDS) and C (detergent mix) yielded highest numbers of peptide identifications (2,495±127 and 2,958±53) and protein groups (298±6 and 324±19) with a low variation between the replicates. Amongst the detergent based extraction methods RapiGest/SP3 (method A) led to a high variation in numbers of identifications between the three replicates performed: 2,105±1503 peptides and 278±115 protein groups. An essential factor influencing the reproducibility is the handling of the tape strips in the extraction procedure. We found handling to be easiest in SDS containing buffers (including the multi detergent mix), in which tape strips were highly flexible, while in comparison RapiGest or buffer conditions apparently increased the rigidity of strips. The increased

Journal Pre-proof flexibility facilitated to place pieces of TS into reaction tubes and covering the whole surface with extraction solution. Beside the conditions tested here, further methods have been described for extraction proteins from TS, e.g. extraction via sodium hydroxide over multiple hours or solubilization of adhesive in organic solvents from strips to remove protein layers.[10,11] We tested these methods in pre-experiments, however, we did not further follow these strategies, as high pH values tend to unspecifically hydrolyze proteins (data not shown).

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As noted above, the analysis of blank TS strips leads to the identification of numerous - also skin related - proteins. Contamination of negative samples with ubiquitously occurring

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keratins is a well-known problem in mass spectrometry analysis, as contaminants can

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originate from either the user or from reagents.[20] However, the number of peptides and protein groups identified using the five different methods A-E differed only slightly, indicating

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that in standard sample procedure the rate of contamination is similar. From these findings,

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independent of the extraction conditions, we strongly recommend to include SP3-supported analysis of blank TS in each study, which is widely missed or not described in existing

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reports. Further, we inspected the LC-MS spectra for potential contaminations deriving e.g.

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from the adhesives or the polymeric matrix of the TS. Overall, we found in several samples polymer signals eluting at the end of the LC-gradient (after 65 minutes), in particular for method C. We conclude that the SP3 based depletion of the detergent mix may not have been quantitative in these cases. However, these signals did not interfere with peptide identifications and the source of contamination was removed after subsequent blank runs. The SDS-concentration used in method B was 4%, which is high compared to most established procedures.[10,21] Hence, we tested three different concentrations - 1%, 2% and 4% (w/v) SDS - on pieces of identical tape strips to evaluate each specific efficiency in triplicates (Suppl. Table 5 & 6). Highest numbers of identified peptides (2839±86) and protein groups (343±17) were achieved for 1%, while for 2% SDS (2751±131/330±12) and 4% SDS (2505±10/291±11) these numbers were lower. However, variation in identification

Journal Pre-proof numbers was increased at lower SDS concentrations compared to 4% SDS condition. We regard this as a minor drawback compared to the gain in higher number of identified peptides. Hence, 1% SDS can be regarded as the optimal concentration for TS extraction using SP3 cleanup. In order to compare these results with previously published methods, we tested the extraction protocol by replacing SP3 clean up with a common protein precipitation protocol while still using low amounts of only slices of single TS. We extracted proteins using in 1 %

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SDS containing lysis buffer and performed an ethanol precipitation. Proteins were then resuspended in identical digestion buffer and then treated as the SP3 samples (Suppl. Table

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7 & 8). By this method 267±13 proteins and 2064±74 peptides could be identified across

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three technical replicates. This demonstrates the SP3 protocol to be the superior compared to precipitation methods, as protein IDs were increased by over 28% and peptide IDs by

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more than 37%, respectively. Also, sample preparation time was clearly reduced compared

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precipitation methods.

In summary, detergent based extraction methods in combination with SP3 sample cleanup

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delivered best results in terms of peptide and protein group identifications. Here, in particular

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the SDS (method B, 1% SDS) and the multi-detergent mixture (method C) mediated procedures showed highest number of identifications, best reproducibility and allow easy handling of the TS.

Application of detergent based extraction/SP3 clean up on samples of different individuals We evaluated the two optimal protein extraction protocols (method B, 1% SDS; and method C) by expanding the number of samples from healthy volunteers to five in order to evaluate potential effects of natural inter-individual biological variation. Both protocols were tested in duplicates per sample using one half of identical tape strips, again including blank tape strips

Journal Pre-proof as negative controls. Figures 3A and B show a moderately increased number of protein and peptide identifications after SDS treatment for most samples, with three exceptions shown on protein level and one on peptide level (Suppl. Table 9-10). Nested Welch t-tests provide a significant increase (p<0.05) of peptide identifications for SDS treated compared to detergent mix samples. Identified proteins in volunteer samples (SDS treated) are nearly identical across all individuals with the majority (190 proteins) identified in all samples (Fig. 3C). Interestingly, samples of volunteer #4 show the smallest amount of identified proteins ,

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missing over 50 proteins identified in other samples. As both extraction methods and TS

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replicates show this trend, we assume this effect to be derived from the volunteers’ sample.

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We performed a string network analysis on all identified proteins in order to validate

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biological reasonableness.[14] The PPI enrichment value for network interaction was smaller than 1.0e-16, thus showing significantly more network interactions than expected. A

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functional enrichment of the network was carried out to obtain GO biological process enrichments. GO term enrichments with an FDR < 1e-45 included cornification, keratinocyte

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differentiation, keratinization, epidermal cell differentiation, skin development, and epidermis

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development (Suppl. Table 11). These highly significant enrichment terms are as expected

obtained by TS.

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for keratinocyte derived proteins and thus highlight the quality of the protein samples

Further improvements of the depth of analysis, and thus the number of identified proteins, could potentially be achieved by the introduction of multidimensional separation schemes after the sample preparation presented here. For example, Newton et al used a combination of SDS-PAGE (for the separation of high abundant keratins) and LC-MS for the identification of 547 proteins in skin biopsies.[22]

Conclusions

Journal Pre-proof To our knowledge, we provide here for the first time a comparative study of different tape strip cell lysis/protein extraction protocols for the analysis of (human) skin samples. A direct comparison with published procedures is hampered by the fact that these procedures either used material of diseased patients, did only rely on total protein determination, worked with samples derived from up to fifteen tape stripes or used elongated lysis times of up to 24h.[8,9] Despite the problems encountered with a direct comparison of the numbers of identified

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peptides and proteins, the introduction of the SP3 sample cleaning allows us to identify a higher number of proteins compared to the earlier presented approaches using samples of

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healthy persons. It should be noted that our analyses were performed with extracts from

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single tape strips, or even only slices of these. This allows for applications aiming to interrogate the protein content in different depths of stratum corneum (stratification) with a

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minimal sample amount.

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Two cell lysis/protein extraction protocols delivered best results: extraction with 1% SDS/SP3 and a multi-detergent mix compatible with SP3. Due to the possibility to work with

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feasible.

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small sample amounts, i.e., single TS, a parallel use of different extraction protocols is

In principle, SP3 sample cleanup could also be replaced by reversed-phase solid phase extraction (SPE). However, we opted for the SP3 method, since it combines several advantages compared to SPE in our workflow: (i) it has been shown to be compatible with different detergents even at elevated concentrations,[13] (ii) it allows to work with relatively high sample volumes and (iii) it is reproducible even at small sample amounts. While the first two aspects hold, at least partial, true for SPE as well, in our experience on SP3-bead digestion has been proven to be more reproducible which was also reported earlier.[23] In negative controls of fresh TS we identified a moderate amount of human proteins, potentially representing contaminations occurring during sample handling, even if we cannot

Journal Pre-proof rule out the presence of these contaminations from vendor-derived TS. While known nonhuman

proteins

are

generally

included

in

contamination

lists

(e.g.

cRAP,

https://www.thegpm.org/crap/), the majority of the remaining proteins are Keratins, which are common contaminations in MS proteomics experiments. While for proteomics analysis of other tissues this might be, at least to some extent, tolerable, in skin focused research we see here a severe issue to be taken into account in the interpretation of the data. Involving blank measurements of the used tape strips is therefore highly recommended.

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We conclude that the SP3 sample cleanup in combination with detergents to be the optimal strategy for TS sample preparation, in particular if analysis will only be performed from single

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TS. As the easiest strategy, SDS was demonstrated to be capable of effectively extracting

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proteins, which could not further be improved by utilizing mixtures of or with other detergents. Further, the SP3 desalting approach is faster than the usually applied protein

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precipitation, which also helps to enhance sample throughput. Thus, the method can be easily applied in a medical/diagnostics environment, providing valuable information to

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decipher molecular mechanisms involved in skin homeostasis and disease development.

Journal Pre-proof Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft (DFG), project TH872/9-1; and the DFG Cluster “Precision Medicine in Inflammation (EXC 22167390884018).

Conflict of Interest

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The authors declare no conflict of interest.

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Journal Pre-proof Figure legends

Fig 1: Protein identifications of tape strip (TS) samples. A: Number of protein identifications after protein extraction from TS using RapiGest, followed by SP3 sample cleanup (left) or RapiGest acid cleavage/precipitation protocol (right). Results of blank control samples are shown in the lower panel. Bars of different columns represent results of different technical replicates. B: Overlap in protein identifications of RapiGest sample extractions using SP3

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protocol or acid cleavage/precipitation (only high confidence protein groups).

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Fig 2: Protein and peptide identification after protein extraction with different extraction buffers from single tape strip (TS) samples; the TS were divided in 5 slices of equal sizes,

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each extracted with the indicated methods. Protein (A) and peptide identifications (B) together with the corresponding control samples (lower panels). Only high confidence

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protein and peptide groups are shown. Bars of different columns represent results of different technical replicates. Extraction methods shown correspond to method A (RapiGest),

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method B (SDS), method C (detergent mix), method D (buffer) and method E (guanidine

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hydrochloride).

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Fig. 3: Comparison of SDS and detergent mix for extraction of proteins from tape strip (TS) samples. Numbers behind TS refers to the tape strip replicate; A-B: Number of protein (A)

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and peptide (B) identification of 10 TS provided by 5 volunteers in duplicates plus blank TS as negative control. C: Intersections between protein identifications across the five volunteers for SDS samples. Numbers of proteins, which were identified in all of the volunteers, as well as in groups of them, are shown. A cut-off of six protein identifications was chosen.

Journal Pre-proof Table 1: Comparison of lysis/extraction methods A - E. Shown are the composition of the lysis/extraction buffer, the extraction conditions and the resulting protein and peptide identification (only high confidence protein and peptide groups). I, II and III (protein//peptide identifications) denote the numbers of replicate.

4% SDS in water; (or 1% SDS)

1% SDS, 1% Triton X100, 1% NP-40, 1% Tween 20, 1% Deoxycholate, 50 mM NaCl, 5 mM EDTA in water

in water

6 M Guanidine hydrochloride in water

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I. 341 II. 304 III. 328 I. 3018 II. 2919 III. 2937

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I. 305 II. 293 III. 297 I. 2628 II. 2480 III. 2376

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96°C for 10 min Sonication for 10 min

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Lysis 56°C for conditions 30 min Sonication for 10 min Protein I. 395 IDs II. 166 III. 273 Peptide I. 3745 IDs II. 794 III. 1776

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0.1% RapiGest in water

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Lysis buffer

Method A Method B Method C Method D Method E 100 mM HEPES (pH 7.5; NaOH), 1x cOmplete Protease inhibitor

I. 119 II. 87 III. 159 I. 428 II. 368 III. 687

I. 79 II. 87 III. 80 I. 566 II. 542 III. 439

Journal Pre-proof Highlights 

Direct comparison of cell lysis and protein extraction methods from identical skin samples Unprecedented numbers of identifications using SP3 sample clean up

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Proteome analysis from single tape strips possible

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Rapid protocol for skin proteome sampling fully compatible with LC-MS

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Journal Pre-proof Biological Significance: Fast and reproducible minimal invasive sampling of human skin proteomes is a major prerequisite for clinical proteomics studies aiming to decipher molecular mechanisms involved in the homeostasis as well as in the development of diseases. By optimization of tape strip sampling, e.g. the introduction of SP3 sample cleanup prior to LC-MS analysis, the presented protocol leads to yet not reported numbers of protein identifications from healthy human skin. Further, due to its efficiency it allows analysis from minimal sample amounts,

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e.g. from single tape strips, while established protocols relied on pooling of multiple tape strips. This provides the opportunity to perform spatially (lateral) resolved proteome analyses

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from different depths of the skin by analysis of consecutive strips.

Journal Pre-proof Conflict of Interest

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The authors declare no conflict of interest.

Figure 1

Figure 2

Figure 3