Extensive differential protein phosphorylation as intraerythrocytic Plasmodium falciparum schizonts develop into extracellular invasive merozoites

Pathology of the most lethal form of malaria is caused by Plasmodium falciparum asexual blood stages and initiated by merozoite invasion of erythrocytes. We present a phosphoproteome analysis of extracellular merozoites revealing 1765 unique phosphorylation sites including 785 sites not previously detected in schizonts. All MS data have been deposited in the ProteomeXchange with identifier PXD001684 ( http://proteomecentral.proteomexchange.org/dataset/PXD001684 ). The observed differential phosphorylation between extra and intraerythrocytic life‐cycle stages was confirmed using both phospho‐site and phospho‐motif specific antibodies and is consistent with the core motif [K/R]xx[pS/pT] being highly represented in merozoite phosphoproteins. Comparative bioinformatic analyses highlighted protein sets and pathways with established roles in invasion. Within the merozoite phosphoprotein interaction network a subnetwork of 119 proteins with potential roles in cellular movement and invasion was identified and suggested that it is coregulated by a further small subnetwork of protein kinase A (PKA), two calcium‐dependent protein kinases (CDPKs), a phosphatidyl inositol kinase (PI3K), and a GCN2‐like elF2‐kinase with a predicted role in translational arrest and associated changes in the ubquitinome. To test this notion experimentally, we examined the overall ubiquitination level in intracellular schizonts versus extracellular merozoites and found it highly upregulated in merozoites. We propose that alterations in the phosphoproteome and ubiquitinome reflect a starvation‐induced translational arrest as intracellular schizonts transform into extracellular merozoites.     

Saliva proteome profiling reveals potential salivary biomarkers for detection of oral cavity squamous cell carcinoma

Oral cavity squamous cell carcinoma (OSCC), which is frequently associated with poor prognosis and mortality, is a leading cause of cancer‐related death worldwide. Discovery of body fluid accessible biomarkers is needed to improve OSCC screening. To this end, we profiled proteomes of saliva from the healthy volunteers, the individuals with oral potentially malignant disorders (OPMD), and the OSCC patients by means of SDS‐PAGE coupled with LC‐MS/MS. In the control, the OPMD, and the OSCC groups, 958, 845, and 1030 salivary proteins were detected, respectively. With spectral counting‐based label‐free quantification, 22 overexpressed salivary proteins were identified in the OSCC group compared with the healthy controls and the OPMD individuals. Among them, resistin (RETN) was subjected to further validation with an independent cohort using ELISA. The data confirmed that the salivary RETN levels in the OSCC patients were significantly higher than that in the healthy or in the OPMD group. Moreover, the elevated levels of salivary RETN were highly correlated with late‐stage primary tumors, advanced overall stage, and lymph‐node metastasis. Our results not only reveal that profiling of saliva proteome is feasible for discovery of OSCC biomarkers, but also identify RETN as a potential salivary biomarker for OSCC detection.     

Analysis of colorectal cancer glyco‐secretome identifies laminin β‐1 (LAMB1) as a potential serological biomarker for colorectal cancer

The high mortality rate in colorectal cancer is mostly ascribed to metastasis, but the only clinical biomarker available for disease monitoring and prognosis is the carcinoembryonic antigen (CEA). However, the prognostic utility of CEA remains controversial. In an effort to identify novel biomarkers that could be potentially translated for clinical use, we collected the secretomes from the colon adenocarcinoma cell line HCT‐116 and its metastatic derivative, E1, using the hollow fiber culture system, and utilized the multilectin affinity chromatography approach to enrich for the secreted glycoproteins (glyco‐secretome). The HCT‐116 and E1 glyco‐secretomes were compared using the label‐free quantitative SWATH‐MS technology, and a total of 149 glycoproteins were differentially secreted in E1 cells. Among these glycoproteins, laminin β‐1 (LAMB1), a glycoprotein not previously known to be secreted in colorectal cancer cells, was observed to be oversecreted in E1 cells. In addition, we showed that LAMB1 levels were significantly higher in colorectal cancer patient serum samples as compared to healthy controls when measured using ELISA. ROC analyses indicated that LAMB1 performed better than CEA at discriminating between colorectal cancer patients from controls. Moreover, the diagnostic performance was further improved when LAMB1 was used in combination with CEA.     

Quantitative proteomics suggests decrease in the secretogranin‐1 cerebrospinal fluid levels during the disease course of multiple sclerosis

Multiple sclerosis (MS) is a chronic inflammatory disease of the CNS with unknown cause. Proteins with different abundance in the cerebrospinal fluid (CSF) from relapsing‐remitting MS (RRMS) patients and neurological controls could give novel insight to the MS pathogenesis and be used to improve diagnosis, predict prognosis and disease course, and guide in therapy decisions. We combined iTRAQ labeling and Orbitrap mass spectrometry to discover proteins with different CSF abundance between six RRMS patients and 18 neurological disease controls. From 777 quantified proteins seven were selected as biomarker candidates, namely chitinase‐3‐like protein 1, secretogranin‐1 (Sg1), cerebellin‐1, neuroserpin, cell surface glycoprotein MUC18, testican‐2 and glutamate receptor 4. An independent sample set of 13 early‐MS patients, 13 RRMS patients and 13 neurological controls was used in a multiple reaction monitoring verification study. We found the intracellular calcium binding protein Sg1 to be increased in early‐MS patients compared to RRMS and neurological controls. Sg1 should be included in further studies to elucidate its role in the early phases of MS pathogenesis and its potential as a biomarker for this disease.     

Minimal sample requirement for highly multiplexed protein quantification in cell lines and tissues by PCT‐SWATH mass spectrometry

The amount of sample available for clinical and biological proteomic research is often limited and thus significantly restricts clinical and translational research. Recently, we have integrated pressure cycling technology (PCT) assisted sample preparation and SWATH‐MS to perform reproducible proteomic quantification of biopsy‐level tissue samples. Here, we further evaluated the minimal sample requirement of the PCT‐SWATH method using various types of samples, including cultured cells (HeLa, K562, and U251, 500 000 to 50 000 cells) and tissue samples (mouse liver, heart, brain, and human kidney, 3–0.2 mg). The data show that as few as 50 000 human cells and 0.2–0.5 mg of wet mouse and human tissues produced peptide samples sufficient for multiple SWATH‐MS analyses at optimal sample load applied to the system. Generally, the reproducibility of the method increased with decreasing tissue sample amounts. The SWATH maps acquired from peptides derived from samples of varying sizes were essentially identical based on the number, type, and quantity of identified peptides. In conclusion, we determined the minimal sample required for optimal PCT‐SWATH analyses, and found smaller sample size achieved higher quantitative accuracy.     

Elevated GAPDH expression is associated with the proliferation and invasion of lung and esophageal squamous cell carcinomas

Glyceraldehyde‐3‐phosphate dehydrogenase, is one of the most investigated housekeeping genes and widely used as an internal control in analysis of gene expression levels. The present study was designed to assess whether GAPDH is associated with cancer cell growth and progression and, therefore may not be a good internal control in cancer research. Our results from clinical tissue studies showed that the levels of GAPDH protein were significantly up‐regulated in lung squamous cell carcinoma tissues, compared with the adjacent normal lung tissues, and this was confirmed by western blotting and immunohistochemistry. GAPDH knockdown by siRNA resulted in significant reductions in proliferation, migration, and invasion of lung squamous carcinoma cells in vitro. In a nude mouse cancer xenograft model, GAPDH knockdown significantly inhibited the cell proliferation and migration/invasion in vivo. In summary, GAPDH may not be an appropriate internal control for gene expression studies, especially in cancer research. The role of GAPDH in cancer development and progression should be further examined in pre‐clinical and clinical studies.     

Long term bed rest with and without vibration exercise countermeasures: effects on human muscle protein dysregulation

The present investigation, the first in the field, was aimed at analyzing differentially, on individual samples, the effects of 55 days of horizontal bed rest, a model for microgravity, on myosin heavy and myosin light chain isoforms distribution (by SDS) and on the proteome (by 2-D DIGE and MS) in the vastus lateralis (VL), a mixed type II/I (～50:50%) head of the quadriceps and in the calf soleus (SOL), a predominantly slow (～35:65%) twitch muscle. Two separate studies were performed on six subjects without (BR) and six with resistive vibration exercise (RVE) countermeasures, respectively. Both VL and SOL underwent in BR decrements of ～15% in cross-sectional area and of ～22% in maximal torque that were prevented by RVE. Myosin heavy chain distribution showed increased type I and decreased type IIA in BR both in VL and in SOL, the opposite with RVE. A substantial downregulation of proteins involved in aerobic metabolism characterized both in SOL and VL in BR. RVE reversed the pattern more in VL than in SOL, whereas proteins involved in anaerobic glycolysis were upregulated. Proteins from the Z-disk region and from costamers were differently dysregulated during bed rest (both BR and RVE), particularly in VL.     

Proteome mining for novel IgE-binding proteins from the German cockroach (Blattella germanica) and allergen profiling of patients

Although cockroaches are known to produce allergens that can cause IgE-mediated hypersensitivity reactions, including perennial rhinitis and asthma, the various cockroach allergens have not yet been fully studied. Many proteins from the German cockroach show high IgE reactivity, but have never been comprehensively characterized. To identify these potential allergens, proteins were separated by 2-DE and IgE-binding proteins were analyzed by nanoLC-MS/MS or N-terminal sequencing analysis. Using a combination of proteomic techniques and bioinformatic allergen database analysis, we identified a total of ten new B. germanica IgE-binding proteins. Of these, aldolase, arginine kinase, enolase, Hsp70, triosephosphate isomerase, and vitellogenin have been reported as allergens in species other than B. germanica. Analysis of the Food Allergy Research and Resource Program allergen database indicated that arginine kinase, enolase, and triosephosphate isomerase showed significant potential cross-reactivity with other related allergens. This study revealed that vitellogenin is an important novel B. germanica allergen. Personalized profiling and reactivity of IgE Abs against the panel of IgE-binding proteins varied between cockroach-allergic individuals. These findings make it possible to monitor the individual IgE reactivity profile of each patient and facilitate personalized immunotherapies for German cockroach allergy disorders.     

Quantitative proteomics study of breast cancer cell lines isolated from a single patient: Discovery of TIMM17A as a marker for breast cancer

The proteins involved in breast cancer initiation and progression are still largely elusive. To gain insights into these processes, we conducted quantitative proteomic analyses with 21T series of breast cell lines, which include a normal, primary tumor and a metastatic tumor that were isolated from a single patient. Stable isotope labeling of amino acid in cell culture followed by LC‐MS/MS analysis was performed and deregulated proteins were identified using statistical analysis. Gene ontology analysis revealed that proteins involved in metabolic processes were the most deregulated in both tumorigenesis and metastasis. Interaction network analysis indicated that ERBB2 signaling played a critical role in tumorigenesis. In addition to known markers such as ERBB2 and E‐cadherin, novel markers, including BRP44L, MTHFD2 and TIMM17A, were found to be overexpressed in 21T breast cancer cells and verified in additional breast cell lines. mRNA expression analysis as well as immunohistochemistry analysis in breast cancer tissues indicated that expression level of TIMM17A was directly correlated with tumor progression, and survival analysis suggested that TIMM17A was a powerful prognosis factor in breast cancer. More interestingly, overexpression and siRNA knockdown experiments indicated an oncogenic activity of TIMM17A in breast cancer. Our study provides a list of potential novel markers for breast cancer tumorigenesis and metastasis using a unique cell model. Further studies on TIMM17A as well as other markers on the list may reveal mechanisms that result in more effective therapeutics for cancer treatment.