Native Proteomics in Discovery Mode Using Size-Exclusion Chromatography–Capillary Zone Electrophoresis–Tandem Mass Spectrometry

Native proteomics aims to characterize complex proteomes under native conditions and ultimately produces a full picture of endogenous protein complexes in cells. It requires novel analytical platforms for high-resolution and liquid-phase separation of protein complexes prior to native mass spectrometry (MS) and MS/MS. In this work, size exclusion chromatography (SEC)-capillary zone electrophoresis (CZE)-MS/MS was developed for native proteomics in discovery mode, resulting in the identification of 144 proteins, 672 proteoforms, and 23 protein complexes from the Escherichia coli proteome. The protein complexes include four protein homodimers, 16 protein-metal complexes, two protein-[2Fe-2S] complexes, and one protein-glutamine complex. Half of them have not been reported in the literature. This work represents the first example of online liquid-phase separation-MS/MS for characterization of a complex proteome under the native condition, offering the proteomics community an efficient and simple platform for native proteomics

The ‘Go’ and ‘Stop’ Mechanisms for Neutrophil Migration

Chemotaxis, or directed cell migration is essential for host defense, embryonic development, axon guidance, and tumor invasion. Chemotactic cells such as blood neutrophils and neutrophil-like, differentiated HL60 cells respond to a gradient of chemoattractant, e.g., fMet-Leu-Phe (fMLP), by adopting polarized morphology (polarization) and crawling up the gradient (directional sensing). How chemotactic cells initiate polarization and orient their polarity precisely toward spatial cues remains unclear. ‘Global inhibitors’ have been postulated to restrict cells from polarizing in random directions, but only permit the direction pointing to spatial cues. we found that moesin, a cross-linker between plasma membrane and actin cytoskeleton, acted as a global inhibitor for neutrophil chemotaxis, which turned-off both ‘frontness’ and ‘backness’ signals (responsible for pseudopod and uropod formation, respectively), and ensured cell's pseudopod correctly orienting toward the source of attractant in a gradient. Removal of moesin not only enhanced small Rho GTPases, Rac, Rho and Cdc42 activities, but also induced cell protrusion in wrong directions, thus abolished cell’s directional sensing. Myosin phosphatase, on the other hand initiated cell migration by breaking down the global inhibition provided by moesin. In order to kill invading bacteria, neutrophils must interpret spatial cues, migrate, and reach target sites. In addition to mechanisms that promote chemotaxis, there are inhibitory mechanisms ensure chemotactic cells to reach correct destinations where they perform bactericidal functions for phagocytes and antigen identification for homing lymphocytes. To date, little is known about the regulatory mechanisms that inhibit cell migration. We show that two mitogen-activated protein kinases played opposing roles in neutrophil trafficking. The extracellular signal-regulated kinase (Erk) potentiated G protein-coupled receptor kinase GRK2 activity and inhibited neutrophil migration, whereas p38 MAPK acted as a non-canonical GRK that phosphorylated the formyl peptide receptor FPR1 and facilitated neutrophil migration by blocking GRK2 function. Therefore, the dynamic balance between Erk and p38 MAPK controls neutrophil “stop” and “go” behaviors, ensuring neutrophils precisely reach their final destination as the first line of host-defense

Additional file 1 of A graph-based approach for proteoform identification and quantification using top-down homogeneous multiplexed tandem mass spectra

Supplementary material. (PDF 162 kb

Characterization of Proteoform Post-Translational Modifications by Top-Down and Bottom-Up Mass Spectrometry in Conjunction with Annotations

Many proteoforms can be produced from a gene due to genetic mutations, alternative splicing, post-translational modifications (PTMs), and other variations. PTMs in proteoforms play critical roles in cell signaling, protein degradation, and other biological processes. Mass spectrometry (MS) is the primary technique for investigating PTMs in proteoforms, and two alternative MS approaches, top-down and bottom-up, have complementary strengths. The combination of the two approaches has the potential to increase the sensitivity and accuracy in PTM identification and characterization. In addition, protein and PTM knowledge bases, such as UniProt, provide valuable information for PTM characterization and verification. Here, we present a software pipeline PTM-TBA (PTM characterization by Top-down and Bottom-up MS and Annotations) for identifying and localizing PTMs in proteoforms by integrating top-down and bottom-up MS as well as PTM annotations. We assessed PTM-TBA using a technical triplicate of bottom-up and top-down MS data of SW480 cells. On average, database search of the top-down MS data identified 2000 mass shifts, 814.5 (40.7%) of which were matched to 11 common PTMs and 423 of which were localized. Of the mass shifts identified by top-down MS, PTM-TBA verified 435 mass shifts using the bottom-up MS data and UniProt annotations

Characterization of Proteoform Post-Translational Modifications by Top-Down and Bottom-Up Mass Spectrometry in Conjunction with Annotations

Many proteoforms can be produced from a gene due to genetic mutations, alternative splicing, post-translational modifications (PTMs), and other variations. PTMs in proteoforms play critical roles in cell signaling, protein degradation, and other biological processes. Mass spectrometry (MS) is the primary technique for investigating PTMs in proteoforms, and two alternative MS approaches, top-down and bottom-up, have complementary strengths. The combination of the two approaches has the potential to increase the sensitivity and accuracy in PTM identification and characterization. In addition, protein and PTM knowledge bases, such as UniProt, provide valuable information for PTM characterization and verification. Here, we present a software pipeline PTM-TBA (PTM characterization by Top-down and Bottom-up MS and Annotations) for identifying and localizing PTMs in proteoforms by integrating top-down and bottom-up MS as well as PTM annotations. We assessed PTM-TBA using a technical triplicate of bottom-up and top-down MS data of SW480 cells. On average, database search of the top-down MS data identified 2000 mass shifts, 814.5 (40.7%) of which were matched to 11 common PTMs and 423 of which were localized. Of the mass shifts identified by top-down MS, PTM-TBA verified 435 mass shifts using the bottom-up MS data and UniProt annotations

Characterization of Proteoform Post-Translational Modifications by Top-Down and Bottom-Up Mass Spectrometry in Conjunction with Annotations

Many proteoforms can be produced from a gene due to genetic mutations, alternative splicing, post-translational modifications (PTMs), and other variations. PTMs in proteoforms play critical roles in cell signaling, protein degradation, and other biological processes. Mass spectrometry (MS) is the primary technique for investigating PTMs in proteoforms, and two alternative MS approaches, top-down and bottom-up, have complementary strengths. The combination of the two approaches has the potential to increase the sensitivity and accuracy in PTM identification and characterization. In addition, protein and PTM knowledge bases, such as UniProt, provide valuable information for PTM characterization and verification. Here, we present a software pipeline PTM-TBA (PTM characterization by Top-down and Bottom-up MS and Annotations) for identifying and localizing PTMs in proteoforms by integrating top-down and bottom-up MS as well as PTM annotations. We assessed PTM-TBA using a technical triplicate of bottom-up and top-down MS data of SW480 cells. On average, database search of the top-down MS data identified 2000 mass shifts, 814.5 (40.7%) of which were matched to 11 common PTMs and 423 of which were localized. Of the mass shifts identified by top-down MS, PTM-TBA verified 435 mass shifts using the bottom-up MS data and UniProt annotations

Characterization of Proteoform Post-Translational Modifications by Top-Down and Bottom-Up Mass Spectrometry in Conjunction with Annotations

Many proteoforms can be produced from a gene due to genetic mutations, alternative splicing, post-translational modifications (PTMs), and other variations. PTMs in proteoforms play critical roles in cell signaling, protein degradation, and other biological processes. Mass spectrometry (MS) is the primary technique for investigating PTMs in proteoforms, and two alternative MS approaches, top-down and bottom-up, have complementary strengths. The combination of the two approaches has the potential to increase the sensitivity and accuracy in PTM identification and characterization. In addition, protein and PTM knowledge bases, such as UniProt, provide valuable information for PTM characterization and verification. Here, we present a software pipeline PTM-TBA (PTM characterization by Top-down and Bottom-up MS and Annotations) for identifying and localizing PTMs in proteoforms by integrating top-down and bottom-up MS as well as PTM annotations. We assessed PTM-TBA using a technical triplicate of bottom-up and top-down MS data of SW480 cells. On average, database search of the top-down MS data identified 2000 mass shifts, 814.5 (40.7%) of which were matched to 11 common PTMs and 423 of which were localized. Of the mass shifts identified by top-down MS, PTM-TBA verified 435 mass shifts using the bottom-up MS data and UniProt annotations

There were significant differences in the survival among three groups (patients with microscopic peritoneal carcinomatosis: group 1; without microscopic peritoneal carcinomatosis: group 2; with macroscopic peritoneal carcinomatosis: group 3).

<p>There were significant differences in the survival among three groups (patients with microscopic peritoneal carcinomatosis: group 1; without microscopic peritoneal carcinomatosis: group 2; with macroscopic peritoneal carcinomatosis: group 3).</p

Characterization of Proteoform Post-Translational Modifications by Top-Down and Bottom-Up Mass Spectrometry in Conjunction with Annotations

Many proteoforms can be produced from a gene due to genetic mutations, alternative splicing, post-translational modifications (PTMs), and other variations. PTMs in proteoforms play critical roles in cell signaling, protein degradation, and other biological processes. Mass spectrometry (MS) is the primary technique for investigating PTMs in proteoforms, and two alternative MS approaches, top-down and bottom-up, have complementary strengths. The combination of the two approaches has the potential to increase the sensitivity and accuracy in PTM identification and characterization. In addition, protein and PTM knowledge bases, such as UniProt, provide valuable information for PTM characterization and verification. Here, we present a software pipeline PTM-TBA (PTM characterization by Top-down and Bottom-up MS and Annotations) for identifying and localizing PTMs in proteoforms by integrating top-down and bottom-up MS as well as PTM annotations. We assessed PTM-TBA using a technical triplicate of bottom-up and top-down MS data of SW480 cells. On average, database search of the top-down MS data identified 2000 mass shifts, 814.5 (40.7%) of which were matched to 11 common PTMs and 423 of which were localized. Of the mass shifts identified by top-down MS, PTM-TBA verified 435 mass shifts using the bottom-up MS data and UniProt annotations

Multivariate analysis on factors in influencing survival.

*<p>MPC microscopic peritoneal carcinomatosis.</p