Project PXD010637

PRIDE Assigned Tags:
Biological Dataset

Summary

Title

Integrated transcriptomic and proteomic analysis of human eccrine sweat glands

Description

The eccrine sweat gland is an exocrine gland that is involved in the secretion of sweat for control of temperature. Malfunction of the sweat glands can result in disorders such as miliaria, hyperhidrosis and bromhidrosis. In addition, inadequate reabsorption of salt from sweat is a major feature of cystic fibrosis. Understanding the transcriptome and proteome of sweat glands is important for understanding the physiology and the role in disease. However, no systematic transcriptome or proteome analysis of sweat glands has yet been reported. To this end, we isolated eccrine sweat glands by microdissecting them from human skin and performed both RNA-seq and proteome analysis. In total, ~138,000 transcripts and ~6,100 proteins were identified. The proteome data of eccrine sweat gland showed enrichment of proteins involved in secretion, reabsorption, and wound healing while the transcriptome data did not show any enrichment for a specific pathway. Importantly, protein level identification of TRPV4 in eccrine sweat gland establishes its importance in re-epithelialization of partial-thickness wound and prevention of dehydration. Furthermore, this study enabled us to identify2 missing proteins. Integration of RNA-seq and proteomic data allowed us to identify 7 peptides from 5 novel genes. Most of the novel proteins were from short open reading frames (sORFs) suggesting that many sORFs still remain to be annotated in the human genome. The peptides mapping to the missing or novel proteins were validated by analyzing synthetic peptides. This study provides the first integrated analysis of the transcriptome and proteome of the human eccrine sweat gland and should become an invaluable resource to biomedical research community for studying sweat glands in physiology and disease.

Sample Processing Protocol

Isolation of eccrine sweat glands Skin tissues were obtained according to approved institutional review board protocol from otherwise healthy adults undergoing unrelated reconstructive surgery at the Department of Plastic and Reconstructive Surgery at the Johns Hopkins University School of Medicine. Eccrine sweat glands were dissected out from the skin dermis as previously described. Briefly, the skin was excised vertically with sharp scissors into small pieces. The cut pieces were bathed in phosphate-buffered Ringer's solution on ice. Neutral red (10 µM) was used to stain the sweat glands. After a few minutes of incubation with neutral red, the sweat glands were viewed through a Zeiss Stemi 2000 dissecting microscope. Fisherbrand fine-pointed forceps were used to dissect eccrine sweat glands from the skin. Connective tissue and fat hanging to the eccrine sweat gland were removed using Fisherbrand dissecting needles with attached holder. Images of the eccrine sweat glands were capturedusing Zeiss microscopy camera, AxioCam lCc 1, and the Axio Vision software (Release 4.8.2). RNA extraction The dissected sweat glands were placed in RNAlater (Qiagen # 76104) immediately after they were dissected out from the skin. The collection tube was centrifuged to remove RNA later solution and Trizol was added to the sample and mixed. The samples were processed in a bead-beater (FastPrep-24, MP) at a setting of 4 m/s for 20 seconds and subsequently placed on ice for five minutes. This was repeated three times. Chloroform was then added to the samples and incubated for five minutes at room temperature. The samples were then centrifuged at 8,000 x g for 5 minutes at 4°C. The aqueous phase was separated from the organic phase containing the DNA and protein. Ethanol was added to the sample for precipitation and immediately applied to RNeasy columns (RNeasy Kit, Qiagen # 74104). The manufacturer’s cleanup procedure was followed except that the RNA sample was treated with the Turbo DNA-Free kit to remove residual DNA contamination (ThermoFisher # AM1907). The RNA was stored at -80°C. The quality of RNA was measured by RNA integrity number (RIN) using Agilent 2100 Bioanalyzer. Preparation of sweat gland proteome To maximize the identification of proteins, the sweat grand proteins were extracted by two different methods - Filter-aided sample prep (FASP) approach and the guanidine hydrochloride (GuHCl) approach. For the FASP approach, sweat glands were sonicated in 50 mM TEAB/4% SDS/10 mM DTT with Halt protease inhibitor cocktail (Thermo Scientific) for 5 min followed by heating at 95°C for 5 min. After cooling, the samples were alkylated with 30 mM iodoacetamide at room temperature for 30 min followed by centrifugation at 17,000 x g for 10 min. SDS in the sample was removed by FAST method.{Wiśniewski, 2009, Universal sample preparation method for proteome analysis} Briefly, the lysate was diluted with 50 mM TEAB/8 M urea and concentrated with centricon with 30 KDa MWCO for 40 min at RT. This buffer exchange step was repeated five times. Sweat gland proteins were digested with Lys-C at room temperature for 3 h followed by further digestion with trypsin overnight. For the GuHCl approach, sweat glands were sonicated for 5 min with 35% amplitude in 8 M GuHCl, 50 mM HEPES, pH 7.0, 10 mm dithiotreitol in the presence of Halt protease inhibitor cocktail (Thermo Scientific) followed by heating at 90°C for 3 min and centrifugation at 16,000 x g for 10 min. The pellet was resuspended in 50 mM TEAB/4% SDS/10 mM DTT and subjected to further protein extraction with barocycler for 60 cycles in which each cycle consisted of 45,000 psi at 95°C for 50 sec and atmospheric pressure at 25oC for 10 sec. The proteins alkylated with 30 mM iodoacetamide at room temperature for 15 min. The proteins were digested with trypsin at the enzyme to protein ratio of 1:50 at 37oC overnight. Following enzyme digestion, the peptides were desalted using Sep-Pak C18 cartridge. The peptides prepared through FASP and GuHCl method were fractionated into 24 fractions by basic pH reversed phase liquid chromatography. Briefly, lyophilized samples were reconstituted in solvent A (10 mM triethylammonium bicarbonate, pH 8.5) and loaded onto XBridge C18, 5 μm 250 × 4.6 mm column (Waters, Milford, MA). Peptides were resolved using a gradient of 3 to 50% solvent B (10 mM triethylammonium bicarbonate in acetonitrile, pH 8.5) over 50 min collecting 96 fractions. The fractions were subsequently concatenated into 24 fractions followed by vacuum drying using SpeedVac. The dried peptides were resuspended in 15 µl 10% FA and the entire amount was injected. The peptides prepared through barocycler were fractionated by SCX StageTip as previously reported.

Data Processing Protocol

RNA-sequencing and data analysis Using 1 µg of total RNA isolated from sweat glands, library preparation and RNA-seq were performed at the Deep Sequencing and Microarray Core Facility at Johns Hopkins University. RNA-seq library was constructed using the TruSeq RNA Sample Prep Kit v2 (Illumina, San Diego, CA, USA), and 200 million paired-end reads were obtained on Illumina’s Next Seq 500 platform. Quality assessment of raw fastq data generate from Illumina was carried out using FastQC v0.11.5. Raw read pairs were subjected to processing using fqtrim v0.9.5 by trimming low quality bases (Q < 20 and length < 35 bp). The human genome version GRCh38 (hg38) was used as reference with gene annotations from GENCODE. Quality processed data was then aligned using HISAT2 v2.0.1 against the human genome. Default parameters of HISAT were used in “--end-to-end” mode. HISAT alignment was then converted to BAM and sorted using samtools v1.3.1 package. Transcript assembly and quantification were carried out using StringTie v1.3.3 package with default parameters. Splice junctions from the alignment data were detected and annotated using RSeQC v2.6.4 package. Extraction of 90 bp sequence from the exon ends of novel splice junctions was carried out followed by translation into peptide sequences for database searching. GTEx pipeline was followed to align against GRCh37 (hg19) version of human genome using GENCODE annotations. Then gene expression values were derived using RNA-SeQC v1.1.8 program and compared against the median RPKM values in GTEx tissues. Data analysis Proteome Discoverer (v 2.1; Thermo Scientific) suite was used for identification and quantitation. The tandem mass spectrometry data were searched using SEQUEST search algorithms against a human RefSeq database (version 70) supplemented with frequently observed contaminants. The search parameters used were as follows: a) trypsin as a proteolytic enzyme (with up to two missed cleavages) b) peptide mass error tolerance of 10 ppm; c) fragment mass error tolerance of 0.02 Da; d) Carbamidomethylation of cysteine (+57.02146 Da) as fixed modification and oxidation of methionine (+15.99492 Da) as variable modifications. Peptides and proteins were filtered at 1% false discovery rate. Protein abundance values were calculated using normalized spectral abundance factor (NSAF). Gene ontology analysis was performed at DAVID. For the identification of novel genes expressed in protein level, transcripts assembled from the RNA-seq data were translated into three frames and a custom database was generated. The database search was conducted in the same way as the search against Reference database except for the enzyme non-specificity on the peptide N-terminal side. For the identification of peptides mapping to novel or partially novel splice junctions, splice junction sequences were extracted with RSeQC software followed by 3 frame translation of the splice junctional sequence spanning 45 nucleotide to the 5’ side and 45 nucleotides to the 3’ side. The database search was conducted in the same way as the search against Reference database. Any peptides that had identical matches in the NR human protein database were removed.

Contact

chanhyun na, Johns Hopkins University
Akhilesh Pandey, Department of Biological Chemistry, McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA ( lab head )

Submission Date

31/07/2018

Publication Date

15/04/2019

Publication

    Na CH, Sharma N, Madugundu AK, Chen R, Atalar Aksit M, Rosson GD, Cutting GR, Pandey A. Integrated transcriptomic and proteomic analysis of human eccrine sweat glands identifies missing and novel proteins. Mol Cell Proteomics. 2019 PubMed: 30979791