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Rapid mass spectrometric conversion of tissue biopsy samples into permanent quantitative digital proteome maps.

Guo T, Kouvonen P, Koh CC, Gillet LC, Wolski WE, Röst HL, Rosenberger G, Collins BC, Blum LC, Gillessen S, Joerger M, Jochum W, Aebersold R - Nat. Med. (2015)

Bottom Line: The method combines pressure cycling technology (PCT) and sequential window acquisition of all theoretical fragment ion spectra (SWATH)-MS.The resulting proteome maps can be analyzed, re-analyzed, compared and mined in silico to detect and quantify specific proteins across multiple samples.From these proteome maps we detected and quantified more than 2,000 proteins with a high degree of reproducibility across all samples.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule (ETH) Zurich, Zurich, Switzerland.

ABSTRACT
Clinical specimens are each inherently unique, limited and nonrenewable. Small samples such as tissue biopsies are often completely consumed after a limited number of analyses. Here we present a method that enables fast and reproducible conversion of a small amount of tissue (approximating the quantity obtained by a biopsy) into a single, permanent digital file representing the mass spectrometry (MS)-measurable proteome of the sample. The method combines pressure cycling technology (PCT) and sequential window acquisition of all theoretical fragment ion spectra (SWATH)-MS. The resulting proteome maps can be analyzed, re-analyzed, compared and mined in silico to detect and quantify specific proteins across multiple samples. We used this method to process and convert 18 biopsy samples from nine patients with renal cell carcinoma into SWATH-MS fragment ion maps. From these proteome maps we detected and quantified more than 2,000 proteins with a high degree of reproducibility across all samples. The measured proteins clearly distinguished tumorous kidney tissues from healthy tissues and differentiated distinct histomorphological kidney cancer subtypes.

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Related in: MedlinePlus

PCT-SWATH method flow chartA batch of 6 biopsy-scale tissues are placed in MicroTubes, and lysed in the barocycler for 60 min. Extracted proteins are reduced and alkylated. The urea concentration is then diluted prior to Lys-c digestion in the barocycler for 45 min. The MicroTubes are then taken out of the barocycler, and the urea concentration is further diluted before trypsin digestion for 90 min under barocycling. The resultant peptides are desalted using C18 cartridges and dried under vacuum. This process can be completed in about 6 hr. The peptide samples are then analyzed using SWATH-MS. The data are analyzed using OpenSWATH and a SWATH assay library.
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Figure 1: PCT-SWATH method flow chartA batch of 6 biopsy-scale tissues are placed in MicroTubes, and lysed in the barocycler for 60 min. Extracted proteins are reduced and alkylated. The urea concentration is then diluted prior to Lys-c digestion in the barocycler for 45 min. The MicroTubes are then taken out of the barocycler, and the urea concentration is further diluted before trypsin digestion for 90 min under barocycling. The resultant peptides are desalted using C18 cartridges and dried under vacuum. This process can be completed in about 6 hr. The peptide samples are then analyzed using SWATH-MS. The data are analyzed using OpenSWATH and a SWATH assay library.

Mentions: First, we optimized a PCT-based protocol that integrates tissue lysis, protein extraction and digestion. The procedure is schematically illustrated in Fig. 1. Briefly, a small piece of wet biopsy tissue, weighing about 1 mg, is placed in a pressure-resistant MicroTube, protein extraction and digestion reagents are added, and the mixture is subjected to pressure cycling. Our method is different from existing preparation of tissue samples for proteomic analysis in that the entire procedure, including tissue lysis, protein extraction and digestion, are integrated as a seamless protocol with minimal sample transfer, permitting analysis of minimal sample amount, and that the reaction solvent volume and incubation time for each step are precisely controlled by the barocycler minimizing technical variation. The resulting peptides are cleaned on reversed-phase columns and are then ready for SWATH-MS.


Rapid mass spectrometric conversion of tissue biopsy samples into permanent quantitative digital proteome maps.

Guo T, Kouvonen P, Koh CC, Gillet LC, Wolski WE, Röst HL, Rosenberger G, Collins BC, Blum LC, Gillessen S, Joerger M, Jochum W, Aebersold R - Nat. Med. (2015)

PCT-SWATH method flow chartA batch of 6 biopsy-scale tissues are placed in MicroTubes, and lysed in the barocycler for 60 min. Extracted proteins are reduced and alkylated. The urea concentration is then diluted prior to Lys-c digestion in the barocycler for 45 min. The MicroTubes are then taken out of the barocycler, and the urea concentration is further diluted before trypsin digestion for 90 min under barocycling. The resultant peptides are desalted using C18 cartridges and dried under vacuum. This process can be completed in about 6 hr. The peptide samples are then analyzed using SWATH-MS. The data are analyzed using OpenSWATH and a SWATH assay library.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4390165&req=5

Figure 1: PCT-SWATH method flow chartA batch of 6 biopsy-scale tissues are placed in MicroTubes, and lysed in the barocycler for 60 min. Extracted proteins are reduced and alkylated. The urea concentration is then diluted prior to Lys-c digestion in the barocycler for 45 min. The MicroTubes are then taken out of the barocycler, and the urea concentration is further diluted before trypsin digestion for 90 min under barocycling. The resultant peptides are desalted using C18 cartridges and dried under vacuum. This process can be completed in about 6 hr. The peptide samples are then analyzed using SWATH-MS. The data are analyzed using OpenSWATH and a SWATH assay library.
Mentions: First, we optimized a PCT-based protocol that integrates tissue lysis, protein extraction and digestion. The procedure is schematically illustrated in Fig. 1. Briefly, a small piece of wet biopsy tissue, weighing about 1 mg, is placed in a pressure-resistant MicroTube, protein extraction and digestion reagents are added, and the mixture is subjected to pressure cycling. Our method is different from existing preparation of tissue samples for proteomic analysis in that the entire procedure, including tissue lysis, protein extraction and digestion, are integrated as a seamless protocol with minimal sample transfer, permitting analysis of minimal sample amount, and that the reaction solvent volume and incubation time for each step are precisely controlled by the barocycler minimizing technical variation. The resulting peptides are cleaned on reversed-phase columns and are then ready for SWATH-MS.

Bottom Line: The method combines pressure cycling technology (PCT) and sequential window acquisition of all theoretical fragment ion spectra (SWATH)-MS.The resulting proteome maps can be analyzed, re-analyzed, compared and mined in silico to detect and quantify specific proteins across multiple samples.From these proteome maps we detected and quantified more than 2,000 proteins with a high degree of reproducibility across all samples.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule (ETH) Zurich, Zurich, Switzerland.

ABSTRACT
Clinical specimens are each inherently unique, limited and nonrenewable. Small samples such as tissue biopsies are often completely consumed after a limited number of analyses. Here we present a method that enables fast and reproducible conversion of a small amount of tissue (approximating the quantity obtained by a biopsy) into a single, permanent digital file representing the mass spectrometry (MS)-measurable proteome of the sample. The method combines pressure cycling technology (PCT) and sequential window acquisition of all theoretical fragment ion spectra (SWATH)-MS. The resulting proteome maps can be analyzed, re-analyzed, compared and mined in silico to detect and quantify specific proteins across multiple samples. We used this method to process and convert 18 biopsy samples from nine patients with renal cell carcinoma into SWATH-MS fragment ion maps. From these proteome maps we detected and quantified more than 2,000 proteins with a high degree of reproducibility across all samples. The measured proteins clearly distinguished tumorous kidney tissues from healthy tissues and differentiated distinct histomorphological kidney cancer subtypes.

Show MeSH
Related in: MedlinePlus