Assessment of protein extraction and digestion efficiency of well-established shotgun protocols for heart proteomics

Lucas Rodrigues-Ribeiro, Marcella Nunes Melo-Braga, Frank Kjeldsen, Diana Paola Gómez-Mendoza*, Thiago Verano-Braga

*Kontaktforfatter for dette arbejde

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Resumé

Ischemic heart disease is the leading cause of deaths worldwide. Thus, understanding the molecular mechanisms underlying disease progression is needed. Due to heart importance and lack of studies evaluating different sample preparation methods for heart proteomics, we compared three well-established protocols in shotgun proteomics using dimethyl label quantitation to allow relative quantitation. The tested methods for the analysis of left ventricle (LV) tissue were: i) in-solution digestion (ISD); ii) on-pellet digestion (OPD); and iii) on-filter digestion (OFD). Protein extraction was done using SDS-containing buffer for OPD and OFD while this step was under urea-containing buffer for ISD. We used an optimized one-step reaction for reduction of disulfide bonds and alkylation of thiol groups in ISD and OPD. Using the same amount of tissue, we observed that OFD and ISD extracted significantly higher amount of protein than OPD. ISD outperformed OFD and OPD in the number of proteins identified. We did not observe significant bias related to physicochemical features of the identified proteins when comparing the three protocols. ISD was more efficient to identify low abundant proteins and yielded more proteins per protocol duration. Thus, we concluded that the optimized ISD suited better for heart proteomics than OFD and OPD.

OriginalsprogEngelsk
TidsskriftAnalytical Biochemistry
Vol/bind578
Sider (fra-til)51-59
ISSN0003-2697
DOI
StatusUdgivet - 1. aug. 2019

Fingeraftryk

Firearms
Proteolysis
Proteins
Buffers
Tissue
Alkylation
Proteomics
Sulfhydryl Compounds
Disulfides
Urea
Labels

Citer dette

Rodrigues-Ribeiro, Lucas ; Melo-Braga, Marcella Nunes ; Kjeldsen, Frank ; Gómez-Mendoza, Diana Paola ; Verano-Braga, Thiago. / Assessment of protein extraction and digestion efficiency of well-established shotgun protocols for heart proteomics. I: Analytical Biochemistry. 2019 ; Bind 578. s. 51-59.
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abstract = "Ischemic heart disease is the leading cause of deaths worldwide. Thus, understanding the molecular mechanisms underlying disease progression is needed. Due to heart importance and lack of studies evaluating different sample preparation methods for heart proteomics, we compared three well-established protocols in shotgun proteomics using dimethyl label quantitation to allow relative quantitation. The tested methods for the analysis of left ventricle (LV) tissue were: i) in-solution digestion (ISD); ii) on-pellet digestion (OPD); and iii) on-filter digestion (OFD). Protein extraction was done using SDS-containing buffer for OPD and OFD while this step was under urea-containing buffer for ISD. We used an optimized one-step reaction for reduction of disulfide bonds and alkylation of thiol groups in ISD and OPD. Using the same amount of tissue, we observed that OFD and ISD extracted significantly higher amount of protein than OPD. ISD outperformed OFD and OPD in the number of proteins identified. We did not observe significant bias related to physicochemical features of the identified proteins when comparing the three protocols. ISD was more efficient to identify low abundant proteins and yielded more proteins per protocol duration. Thus, we concluded that the optimized ISD suited better for heart proteomics than OFD and OPD.",
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Assessment of protein extraction and digestion efficiency of well-established shotgun protocols for heart proteomics. / Rodrigues-Ribeiro, Lucas; Melo-Braga, Marcella Nunes; Kjeldsen, Frank; Gómez-Mendoza, Diana Paola; Verano-Braga, Thiago.

I: Analytical Biochemistry, Bind 578, 01.08.2019, s. 51-59.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - Assessment of protein extraction and digestion efficiency of well-established shotgun protocols for heart proteomics

AU - Rodrigues-Ribeiro, Lucas

AU - Melo-Braga, Marcella Nunes

AU - Kjeldsen, Frank

AU - Gómez-Mendoza, Diana Paola

AU - Verano-Braga, Thiago

N1 - Copyright © 2019 Elsevier Inc. All rights reserved.

PY - 2019/8/1

Y1 - 2019/8/1

N2 - Ischemic heart disease is the leading cause of deaths worldwide. Thus, understanding the molecular mechanisms underlying disease progression is needed. Due to heart importance and lack of studies evaluating different sample preparation methods for heart proteomics, we compared three well-established protocols in shotgun proteomics using dimethyl label quantitation to allow relative quantitation. The tested methods for the analysis of left ventricle (LV) tissue were: i) in-solution digestion (ISD); ii) on-pellet digestion (OPD); and iii) on-filter digestion (OFD). Protein extraction was done using SDS-containing buffer for OPD and OFD while this step was under urea-containing buffer for ISD. We used an optimized one-step reaction for reduction of disulfide bonds and alkylation of thiol groups in ISD and OPD. Using the same amount of tissue, we observed that OFD and ISD extracted significantly higher amount of protein than OPD. ISD outperformed OFD and OPD in the number of proteins identified. We did not observe significant bias related to physicochemical features of the identified proteins when comparing the three protocols. ISD was more efficient to identify low abundant proteins and yielded more proteins per protocol duration. Thus, we concluded that the optimized ISD suited better for heart proteomics than OFD and OPD.

AB - Ischemic heart disease is the leading cause of deaths worldwide. Thus, understanding the molecular mechanisms underlying disease progression is needed. Due to heart importance and lack of studies evaluating different sample preparation methods for heart proteomics, we compared three well-established protocols in shotgun proteomics using dimethyl label quantitation to allow relative quantitation. The tested methods for the analysis of left ventricle (LV) tissue were: i) in-solution digestion (ISD); ii) on-pellet digestion (OPD); and iii) on-filter digestion (OFD). Protein extraction was done using SDS-containing buffer for OPD and OFD while this step was under urea-containing buffer for ISD. We used an optimized one-step reaction for reduction of disulfide bonds and alkylation of thiol groups in ISD and OPD. Using the same amount of tissue, we observed that OFD and ISD extracted significantly higher amount of protein than OPD. ISD outperformed OFD and OPD in the number of proteins identified. We did not observe significant bias related to physicochemical features of the identified proteins when comparing the three protocols. ISD was more efficient to identify low abundant proteins and yielded more proteins per protocol duration. Thus, we concluded that the optimized ISD suited better for heart proteomics than OFD and OPD.

KW - Dimethyl labeling

KW - Heart

KW - Protein extraction

KW - Sample preparation

KW - Shotgun proteomics

U2 - 10.1016/j.ab.2019.05.006

DO - 10.1016/j.ab.2019.05.006

M3 - Journal article

VL - 578

SP - 51

EP - 59

JO - Analytical Biochemistry

JF - Analytical Biochemistry

SN - 0003-2697

ER -