TY - JOUR
T1 - A meta-analysis of affinity purification-mass spectrometry experimental systems used to identify eukaryotic and chlamydial proteins at the Chlamydia trachomatis inclusion membrane
AU - Olson, Macy G.
AU - Ouellette, Scot P.
AU - Rucks, Elizabeth A.
N1 - Funding Information:
This work was supported by UNMC start-up funds for E.A. Rucks and S.P. Ouellette and supported by an R01AI114670-01A1 awarded to E.A. Rucks and an R35GM124798-01 awarded to S.P. Ouellette. Macy. G. Olson is partially supported by a University of Nebraska Medical Center Program of Excellence. Assistantship.
Funding Information:
This work was supported by UNMC start-up funds for E.A. Rucks and S.P. Ouellette and supported by an R01AI114670-01A1 awarded to E.A. Rucks and an R35GM124798-01 awarded to S.P. Ouellette. Macy. G. Olson is partially supported by a University of Nebraska Medical Center Program of Excellence. Assistantship.
Publisher Copyright:
© 2019
PY - 2020/2/10
Y1 - 2020/2/10
N2 - The obligate intracellular bacterial pathogen, Chlamydia trachomatis, develops within a membrane-bound vacuole termed the inclusion. Affinity purification-mass spectrometry (AP-MS) experiments to study the interactions that occur at the chlamydial inclusion membrane have been performed and, more recently, combined with advances in C. trachomatis genetics. However, each of the four AP-MS published reports used either different experimental approaches or statistical tools to identify proteins that localize at the inclusion. We critically analyzed each experimental approach and performed a meta-analysis of the reported statistically significant proteins for each study, finding that only a few eukaryotic proteins were commonly identified between all four experimental approaches. The two similarly conducted in vivo labeling studies were compared using the same statistical analysis tool, Significance Analysis of INTeractome (SAINT), which revealed a disparity in the number of significant proteins identified by the original analysis. We further examined methods to identify potential background contaminant proteins that remain after statistical analysis. Overall, this meta-analysis highlights the importance of carefully controlling and analyzing the AP-MS data so that pertinent information can be obtained from these various AP-MS experimental approaches. This study provides important guidelines and considerations for using this methodology to study intracellular pathogens residing within a membrane-bound compartment. Significance: Chlamydia trachomatis, an obligate intracellular pathogen, grows within a membrane-bound vacuole termed the inclusion. The inclusion is studded with bacterial membrane proteins that likely orchestrate numerous interactions with the host cell. Although maintenance of the intracellular niche is vital, an understanding of the host-pathogen interactions that occur at the inclusion membrane is limited by the difficulty in purifying membrane protein fractions from infected host cells. The experimental procedures necessary to solubilize hydrophobic proteins fail to maintain transient protein-protein interactions. Advances in C. trachomatis genetics has allowed us and others to use various experimental approaches in combination with affinity purification mass spectrometry (AP-MS) to study the interactions that occur at the chlamydial vacuolar, or inclusion, membrane. For the first time, two groups have published AP-MS studies using the same tool, the ascorbate peroxidase proximity labeling system (APEX2), which overcomes past experimental limitations because membrane protein interactions are labeled in vivo in the context of infection. The utility of this system is highlighted by its ability to study chlamydial type III secreted inclusion membrane protein (Inc) interactions. Incs act as the mediators of host-pathogen interactions at the inclusion during C. trachomatis infection. When carefully controlled and analyzed, the data obtained can yield copious amounts of useful information. Here, we critically analyzed four previously published studies, including statistical analysis of AP-MS datasets related to Chlamydia-host interactions, to contextualize the data and to identify the best practices in interpreting these types of complex outputs.
AB - The obligate intracellular bacterial pathogen, Chlamydia trachomatis, develops within a membrane-bound vacuole termed the inclusion. Affinity purification-mass spectrometry (AP-MS) experiments to study the interactions that occur at the chlamydial inclusion membrane have been performed and, more recently, combined with advances in C. trachomatis genetics. However, each of the four AP-MS published reports used either different experimental approaches or statistical tools to identify proteins that localize at the inclusion. We critically analyzed each experimental approach and performed a meta-analysis of the reported statistically significant proteins for each study, finding that only a few eukaryotic proteins were commonly identified between all four experimental approaches. The two similarly conducted in vivo labeling studies were compared using the same statistical analysis tool, Significance Analysis of INTeractome (SAINT), which revealed a disparity in the number of significant proteins identified by the original analysis. We further examined methods to identify potential background contaminant proteins that remain after statistical analysis. Overall, this meta-analysis highlights the importance of carefully controlling and analyzing the AP-MS data so that pertinent information can be obtained from these various AP-MS experimental approaches. This study provides important guidelines and considerations for using this methodology to study intracellular pathogens residing within a membrane-bound compartment. Significance: Chlamydia trachomatis, an obligate intracellular pathogen, grows within a membrane-bound vacuole termed the inclusion. The inclusion is studded with bacterial membrane proteins that likely orchestrate numerous interactions with the host cell. Although maintenance of the intracellular niche is vital, an understanding of the host-pathogen interactions that occur at the inclusion membrane is limited by the difficulty in purifying membrane protein fractions from infected host cells. The experimental procedures necessary to solubilize hydrophobic proteins fail to maintain transient protein-protein interactions. Advances in C. trachomatis genetics has allowed us and others to use various experimental approaches in combination with affinity purification mass spectrometry (AP-MS) to study the interactions that occur at the chlamydial vacuolar, or inclusion, membrane. For the first time, two groups have published AP-MS studies using the same tool, the ascorbate peroxidase proximity labeling system (APEX2), which overcomes past experimental limitations because membrane protein interactions are labeled in vivo in the context of infection. The utility of this system is highlighted by its ability to study chlamydial type III secreted inclusion membrane protein (Inc) interactions. Incs act as the mediators of host-pathogen interactions at the inclusion during C. trachomatis infection. When carefully controlled and analyzed, the data obtained can yield copious amounts of useful information. Here, we critically analyzed four previously published studies, including statistical analysis of AP-MS datasets related to Chlamydia-host interactions, to contextualize the data and to identify the best practices in interpreting these types of complex outputs.
KW - APEX2
KW - Affinity purification-mass spectrometry
KW - Bacteria-containing vacuole or inclusion
KW - Chlamydia trachomatis
KW - Inc
KW - SAINT
UR - http://www.scopus.com/inward/record.url?scp=85075763219&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85075763219&partnerID=8YFLogxK
U2 - 10.1016/j.jprot.2019.103595
DO - 10.1016/j.jprot.2019.103595
M3 - Article
C2 - 31760040
AN - SCOPUS:85075763219
SN - 1874-3919
VL - 212
JO - Journal of Proteomics
JF - Journal of Proteomics
M1 - 103595
ER -