Fully Automated Four-Column Capillary LC−MS System for Maximizing Throughput in Proteomic Analyses

Livesay, Eric A.; Tang, Keqi; Taylor, Beverley K.; Buschbach, Michael A.; Hopkins, Derek F.; LaMarche, Brian L.; Zhao, Rui; Shen, Yufeng; Orton, Daniel J.; Moore, Ronald J.; Kelly, Ryan; Udseth, Harold R.; Smith, Richard D.

doi:10.1021/ac701727r

Cited by 132 publications

(161 citation statements)

References 25 publications

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“…reversed-phase capillary column using 3 mm C18 particles (Phenomenex, Torrance, CA). The HPLC system consisted of a custom configuration of 100 ml Isco Model 100DM syringe pumps (Isco, Lincoln, NE), two-position Valco valves (Valco Instruments Co., Houston, TX), and a PAL autosampler (Leap Technologies, Carrboro, NC) that allowed fully automated sample analysis across four HPLC columns (13). The system was operated at a constant pressure of 10,000 psi over 3 h with an exponential gradient starting with 100% of mobile phase A (0.1% (v/v) formic acid in water) to 60% (v/v) of mobile phase B (0.1% (v/v) formic acid in acetonitrile).…”

Section: Methodsmentioning

confidence: 99%

Global Proteomic Analysis Reveals an Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model Cyanobacterium

Liberton

Saha

Jacobs

et al. 2016

Molecular & Cellular Proteomics

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Cyanobacteria are photosynthetic microbes with highly differentiated membrane systems. These organisms contain an outer membrane, plasma membrane, and an internal system of thylakoid membranes where the photosynthetic and respiratory machinery are found. This existence of compartmentalization and differentiation of membrane systems poses a number of challenges for cyanobacterial cells in terms of organization and distribution of proteins to the correct membrane system. Proteomics studies have long sought to identify the components of the different membrane systems in cyanobacteria, and to date about 450 different proteins have been attributed to either the plasma membrane or thylakoid membrane. Given the complexity of these membranes, many more proteins remain to be identified, and a comprehensive catalogue of plasma membrane and thylakoid membrane proteins is needed. Here we describe the identification of 635 differentially localized proteins in Synechocystis sp. PCC 6803 by quantitative iTRAQ isobaric labeling; of these, 459 proteins were localized to the plasma membrane and 176 were localized to the thylakoid membrane. Surprisingly, we found over 2.5 times the number of unique proteins identified in the plasma membrane compared with the thylakoid membrane. This suggests that the protein composition of the thylakoid membrane is more homogeneous than the plasma membrane, consistent with the role of the plasma membrane in diverse cellular processes including protein trafficking and nutrient import, compared with a more specialized role for the thylakoid membrane in cellular energetics. Thus, our data clearly define the two membrane systems with distinct functions. Overall, the protein compositions of the Synechocystis 6803 plasma membrane and thylakoid membrane are quite similar to that of the plasma membrane of Escherichia coli and thylakoid membrane of Arabidopsis chloroplasts, respectively. Synechocystis 6803 can therefore be described as a Gram-negative bacterium with an additional internal membrane system that fulfills the energetic requirements of the cell. Photosynthetic microbes such as the cyanobacterium Synechocystis sp. PCC 6803 convert light to cellular energy, an ability that makes these organisms of particular interest in renewable energy studies. Cyanobacteria typically have a Gram-negative-type cell envelope consisting of a plasma membrane (PM) 1 , peptidoglycan layer, and outer membrane. These microbes also have an internal thylakoid membrane (TM) system where the protein complexes of the photosynthetic and respiratory electron transfer chains function. The presence of these differentiated membrane systems makes cyanobacteria more complex than other Gram-negative bacteria. There is considerable interest in understanding the roles of the membrane systems and their relation with each other. Our studies using electron tomography revealed that the TM in the cyanobacterium Cyanothece sp. ATCC 51142 forms a complicated network of membranes that enclose a single lumenal space (1). Several studies have ...

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Section: Methodsmentioning

confidence: 99%

Global Proteomic Analysis Reveals an Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model Cyanobacterium

Liberton

Saha

Jacobs

et al. 2016

Molecular & Cellular Proteomics

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“…Then, the samples were 4-fold diluted with 25 mM NH4HCO3 and 1 mM CaCl2 and digested overnight with a 1:20 (mass/mass) trypsin-to-protein ratio. The digested peptides were desalted with C18 cartridges and dried in a vacuum centrifuge before being separated in capillary columns (75 μm × 65 cm capillary [Polymicro] packed with 3-μm C18 particles [Phenomenex]) connected to a custom-made 4-column liquid chromatography LC system (77) or a longer capillary column (75 μm × 100 cm) connected to a nanoAcquity system (Waters). Eluting peptides were analyzed directly in a linear ion-trap orbitrap mass spectrometer (LTQ Orbitrap XL, Thermo Scientific).…”

Section: Methodsmentioning

confidence: 99%

Cytomegalovirus pp65 limits dissemination but is dispensable for persistence

et al. 2014

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The most abundantly produced virion protein in human cytomegalovirus (HCMV) is the immunodominant phosphoprotein 65 (pp65), which is frequently included in CMV vaccines. Although it is nonessential for in vitro CMV growth, pp65 displays immunomodulatory functions that support a potential role in primary and/or persistent infection. To determine the contribution of pp65 to CMV infection and immunity, we generated a rhesus CMV lacking both pp65 orthologs (RhCMVΔpp65ab). While deletion of pp65ab slightly reduced growth in vitro and increased defective particle formation, the protein composition of secreted virions was largely unchanged. Interestingly, pp65 was not required for primary and persistent infection in animals. Immune responses induced by RhCMVΔpp65ab did not prevent reinfection with rhesus CMV; however, reinfection with RhCMVΔUS2-11, which lacks viral-encoded MHC-I antigen presentation inhibitors, was prevented. Unexpectedly, induction of pp65b-specific T cells alone did not protect against RhCMVΔUS2-11 challenge, suggesting that T cells targeting multiple CMV antigens are required for protection. However, pp65-specific immunity was crucial for controlling viral dissemination during primary infection, as indicated by the marked increase of RhCMVΔpp65ab genome copies in CMV-naive, but not CMV-immune, animals. Our data provide rationale for inclusion of pp65 into CMV vaccines but also demonstrate that pp65-induced T cell responses alone do not recapitulate the protective effect of natural infection.

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“…The desalted peptides were separated with an automated reverse-phase capillary LC system designed in house (26). Eluate from the LC was directly electrosprayed into an LTQ-Orbitrap mass spectrometer (Thermo Fisher, San Jose, CA) by using an electrospray ionization interface manufactured in house.…”

Section: Methodsmentioning

confidence: 99%

Proteomic Investigation of the Time Course Responses of RAW 264.7 Macrophages to Infection with Salmonella enterica

et al. 2009

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To investigate the extent to which macrophages respond to Salmonella infection, we infected RAW 264.7 macrophages with Salmonella enterica serotype Typhimurium and analyzed macrophage proteins at various time points following infection by using a global proteomic approach. A total of 1,006 macrophage and 115 Salmonella proteins were identified with high confidence. Most of the Salmonella proteins were observed in the late stage of the infection time course, which is consistent with the fact that the bacterial cells proliferate inside RAW 264.7 macrophages. The peptide abundances of most of the identified macrophage proteins remained relatively constant over the time course of infection. Compared to those of the control, the peptide abundances of 244 macrophage proteins (i.e., 24% of the total identified macrophage proteins) changed significantly after infection. The functions of these Salmonella-affected macrophage proteins were diverse, including production of antibacterial nitric oxide (i.e., inducible nitric oxide synthase), production of prostaglandin H 2 (i.e., cyclooxygenase 2), and regulation of intracellular traffic (e.g., sorting nexin 5 [SNX5], SNX6, and SNX9). Diverse functions of the Salmonella-affected macrophage proteins demonstrate a global macrophage response to Salmonella infection. Western blot analysis not only confirmed the proteomic results for a selected set of proteins but also revealed that (i) the protein abundance of mitochondrial superoxide dismutase increased following macrophage infection, indicating an infection-induced oxidative stress in mitochondria, and (ii) in contrast to infection of macrophages by wild-type Salmonella, infection by the sopB deletion mutant had no negative impact on the abundance of SNX6, suggesting a role for SopB in regulating the abundance of SNX6.

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Fully Automated Four-Column Capillary LC−MS System for Maximizing Throughput in Proteomic Analyses

Cited by 132 publications

References 25 publications

Global Proteomic Analysis Reveals an Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model Cyanobacterium

Global Proteomic Analysis Reveals an Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model Cyanobacterium

Cytomegalovirus pp65 limits dissemination but is dispensable for persistence

Proteomic Investigation of the Time Course Responses of RAW 264.7 Macrophages to Infection with Salmonella enterica

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