From 1034020dd4520f005b5b05b38346b52c797eb01e Mon Sep 17 00:00:00 2001 From: Pia Saake <psaake@uni-koeln.de> Date: Wed, 16 Oct 2024 20:58:53 +0200 Subject: [PATCH] add microscopy and proteome analysis --- .gitattributes | 3 + .../README.md} | 0 .../dataset/.gitkeep | 0 .../dataset/Figure 1.png | 3 + .../isa.assay.xlsx | Bin 0 -> 6919 bytes .../protocols/.gitkeep | 0 ...ITC488 labeling and confocal microscopy.md | 14 ++++ .../README.md | 4 ++ .../dataset/Figure 2.pdf | 3 + .../dataset/Figure 2.png | 3 + .../dataset/TableS1_all_proteins.xlsx | 3 + .../dataset/TableS2_SP_proteins.xlsx | 3 + .../isa.assay.xlsx | Bin 6933 -> 7066 bytes .../protocols/LC-MSMS.md | 62 ++++++++++++++++++ ...dica EPS matrix CW and culture filtrate.md | 43 ++++++++++++ 15 files changed, 141 insertions(+) rename assays/{Proteome analysis of S indica EPS matrix CW and cullture filtrate/protocols/Proteome analysis of S. indica EPS matrix, CW, and culture filtrate => Microscopy of S indica EPS matrix and 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for SiWSC3 labeling: 20-min incubation +time at 20°C with half of the recommended concentration. +The reaction was stopped by adding 1-M TRIS (pH 7.5) to a +final concentration of 50 mM and then dialyzed overnight +against 3 L of Milli-Q water. Modifications to the standard +labeling protocol were necessary because over labeling re- +duced the ability of the SiWSC3 to bind to its substrate +(Wawra et al., 2019). \ No newline at end of file diff --git a/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/README.md b/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/README.md index e69de29..25fcee5 100644 --- a/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/README.md +++ b/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/README.md @@ -0,0 +1,4 @@ +Data is visualized in Figure 2 A-B. + +Raw data is provided in Table S1 for A (total proteins) and Table S2 for A (Proteins with predicted signal peptide) + diff --git a/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/dataset/Figure 2.pdf b/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/dataset/Figure 2.pdf new file mode 100644 index 0000000..22aa526 --- /dev/null +++ b/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/dataset/Figure 2.pdf @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:31b1579e2e0e45556a59fc149e38abcc51107f610eef2e97215bf74cf605f536 +size 1814993 diff --git a/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/dataset/Figure 2.png b/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/dataset/Figure 2.png new file mode 100644 index 0000000..32168b0 --- /dev/null +++ b/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/dataset/Figure 2.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:16f3e1ef14fc39296faf943869230903c21985df1f9c1ceebcbc44aba158a892 +size 1084936 diff --git a/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/dataset/TableS1_all_proteins.xlsx b/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/dataset/TableS1_all_proteins.xlsx new file mode 100644 index 0000000..73fdc1d --- /dev/null +++ b/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/dataset/TableS1_all_proteins.xlsx @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:a2e57f204ffc1eaeaa3a9ffe48fdc5dfe80fd2037670f4a6959440bb9d28f855 +size 663068 diff --git a/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/dataset/TableS2_SP_proteins.xlsx b/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/dataset/TableS2_SP_proteins.xlsx new file mode 100644 index 0000000..b6a8b65 --- /dev/null +++ b/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/dataset/TableS2_SP_proteins.xlsx @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:c09d42a7c863366af8180052c1f0e8eae2c1f9477a7959895dc9af86e2058855 +size 85809 diff --git a/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/isa.assay.xlsx b/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/isa.assay.xlsx index f75b32f9697784b2e5a47fde0d3b559c36cea043..09d97d00925201c93bda5c8c0b2da50e769108c6 100644 GIT binary patch delta 2076 zcmZ8ic{tSDAN~%R$TG5xVJwreQ`t#~p&27fG-}dCHMV4TC2?m&W63tgzNSpqjP2S+ zcubZSTdrO9igrtdNV$Gvy8WKVJ^#FCdCz;!^PJCl-%R~H13?EH?tKRU0N@2+*$y>= zaUgCsQnNvbU94*XL4Hm=<C5hp4DyPJx&?b&4LP!BW1Tp+>`$WpK2;(1PP~oIYr8@& z0MI``_1kxdI3+LqbZ$dcV^&%qfofR$Tf=RJ?PMwaW4+hSprk9({Fuq*;T4Ol_)?~` ztj0IP%^A?f{@ST=X2MqsX?io9Vx4qoFbLP<pMTHI9%5i42J3B3*JFqpMTmlDB#LXH zv|?M$*koWJ7d|@nRXs_t+N!tDa{Vs1JuS3XBPW7*+qRlVedZ3cT{q?J1CLr1x&up$ zEn#4TYn&s;6Z>Tm=XLumw(e)O8V#QR=ir6jwbiPJFBwaq8cK3~fKcMytZJ6OF>T_0 zw9mtI*t{}RXZEE_cY`*gR<v!~W^KE`so;{lI9~N4I1ZOuUltsScHYR4zd)#3jj{O> zmx3jpuKDe<+-NTp(b#ay1nW>5#-tQ(Dt)~RI-ByUm-#m%$nWql?i*8r+!^s&#kwCc z)>nII_=DojOy$3NO^g>6@RaDF&#_8rx@K#wF+u}`wpnNpdBciq6iGU^TK4qb&iv_A zbpP?UEgMmxhGV+Ov5|S(>Pz2P6M+?YOhqp7sZAAo^ewOgNw615ZIpcT3>6-d+6aH6 zX%Wt!nk}@Nh45`z^HpM<pXOJFrDTyUZKC8`6T@B>2%!~%(%LS`8962*Pt~!!t+j8K zns@Qfq=;$df)P=C>6Bwib@HXvy4sFCNR00zs5Ff3DS|Y3^)WdG&Ol;<9%aL2h^Ik# zHu>TpWj?3W9OGsZYH6a2F&Q}vkvR<!bcC#rqN+z@TEEwOxqL2sU%7Z{B)%uwU19C> za@daZ`Z9#!eXjzy)XjKFmtSk=f}O!mP@_|)i#wLLFg|>I%~1Q42wJ7*P=~ws&b_w5 ziW_`Taa*tVk>*Y>I#&C!rZ0#`2ULXOL@UlIE>X8MZCqN$NjiVqr=Y-_KNi&g;o4Kn zc)ZPJ%M}m+pm_j5jQusjuO12Uz8LI5a0(6f@%IWj9PW2D^Gv&c{t4mM4fT@+lPE#! 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Protein-containing +bands from Coomassie-stained gels were prepared for mass +spectrometric analysis as described elsewhere (Poschmann +et al., 2014). Briefly, bands were destained and the proteins +were reduced with dithiothreitol and alkylated with iodoace- +tamide and subjected to tryptic digestion. The resulting pep- +tides were extracted and reconstituted in 0.1% (v/v) +trifluoroacetic acid in water. Peptides were separated on an +Ultimate 3000 Rapid Separation Liquid Chromatography sys- +tem (Thermo Fisher Scientific) on a 25-cm length C18 col- +umn using a 1-h gradient and subsequently analyzed by a +QExactive Plus mass spectrometer (Thermo Fisher Scientific) +as described with minor modifications (Poschmann et al., +2014). First, survey scans were carried out at a resolution of +140,000 and up to 22- and 3-fold charged precursors se- +lected by the quadrupole (4 m/z isolation window), frag- +mented by higher energy collisional dissociation and +fragment spectra recorded at a resolution of 17,500. Mass +spectrometric data were further processed by MaxQuant +1.6.12.0 (Max-Planck Institute for Biochemistry, Planegg, +Germany) with standard parameters if not otherwise stated. +Label-free quantification, “match between runs†and iBAQ +quantification were enabled. Searches were carried out based +on S. indica reference protein entries (UP000007148), down- +loaded on 15 May 2020 from the UniProt Knowledge Base. +Carbamidomethylation at cysteines was considered as fixed +and methionine oxidation and proteins N-terminal acetyla- +tion as variable modifications. Peptides and proteins were +accepted at a false discovery rate of 1% and only proteins +further considered were identified with at least two different +peptides. The identified proteins were grouped into families +using the Pfam database (Mistry et al., 2021). The mass spec- +trometry proteomics data have been deposited to the +ProteomeXchange Consortium via the PRIDE partner reposi- +tory with the dataset identifier PXD025640. + +## Proteome analysis of S. indica EPS matrix, CW, and culture filtrate + +Further calculations were done on label-free quantification +(LFQ) intensity values. Only proteins were considered show- +ing at least two valid intensity values at least in one group. +LFQ intensity values were log2 transformed and missing val- +ues imputed with values drawn from a downshifted normal +distribution (width 0.3, downshift 1.8) before statistical and +enrichment analysis. Statistical analysis was done for selected +protein groups using the significance analysis of microarrays +method (Tusher et al., 2001;5% false discovery +rate, S0 = 0.1) and Student’s t test. To identify proteins contain- +ing certain domains showing a higher abundance in the EPS +matrix, we performed an annotation enrichment analysis +(Cox and Mann, 2012) based on the differences of mean val- +ues of log2 transformed LFQ intensities. +The percentage relative abundance of signal peptide con- +taining proteins detected in the three components (EPS ma- +trix, CW, and culture filtrate) was calculated using LFQ +intensities. \ No newline at end of file diff --git a/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/protocols/Protein isolation of S indica EPS matrix CW and culture filtrate.md b/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/protocols/Protein isolation of S indica EPS matrix CW and culture filtrate.md new file mode 100644 index 0000000..40d8316 --- /dev/null +++ b/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/protocols/Protein isolation of S indica EPS matrix CW and culture filtrate.md @@ -0,0 +1,43 @@ +# Protein isolation of S. indica EPS matrix, CW, and culture filtrate + +The proteins were isolated from the EPS matrix, CW, and +the culture filtrate obtained from axenic cultures of S. indica +strain expressing GFP grown in different media (CM, YPD, +and TSB). + +## Protein isolation from the CW + +Mycelium collected from the S. indica GFP strain was +ground in liquid N2 and resuspended in PBS buffer contain- +ing 1-mM PMSF and 1% (v/v) NP-40 using an ULTRA- +TURRAX (IKA, Staufen, Germany). The resuspended mixture +was incubated at 4°C in a rotating wheel for 30 min. The +pellet obtained after centrifugation at 8,000g for 15 min at +4°C was resuspended in PBS buffer containing 1-mM PMSF +and 0.1% (v/v) IGEPAL using an ULTRA-TURRAX. The pellet +obtained after centrifugation at 8,000g for 15 min at 4°C +was washed three times with Milli-Q water. Finally, the pel- +let was resuspended in Laemmli buffer containing 8-M urea, +2-M thiourea, and b-mercaptoethanol and boiled at 95°C +for 10 min. + +## Protein isolation from the EPS matrix + +The EPS matrix obtained from the culture media by cryoge- +lation was washed four times with Milli-Q water and was di- +rectly boiled in Laemmli buffer containing 8-M urea, 2-M +thiourea, and b-mercaptoethanol at 95°C for 10 min. + +## Protein isolation from the culture filtrate + +The culture supernatant left over from the EPS matrix isola- +tion step was first filtered using a Whatman filter paper and +then using a 0.22-mM syringe filter. Approximately 30 mL of +EPS matrix depleted culture supernatant was treated with 5 +mL of 95% (v/v) trichloroacetic acid and incubated over- +night at 4°C. The precipitated proteins were collected by +centrifugation (10,000g) for 1 h at 4°C. The isolated proteins +were washed at least three times with 100% (v/v) acetone. +The dried protein pellet was resuspended in Laemmli buffer +containing 8-M urea, 2-M thiourea, and b-mercaptoethanol +andboiledat 95°C for 10 min. \ No newline at end of file -- GitLab