Notes: These authors evaluated three commonly used cell lysis buffers based on the number of proteins and peptides identified in shotgun UPLC-MS analysis. Detergents used included urea, SDC, and ProteaseMAX™ Surfactant. The proteins were digested with trypsin. The buffer that included ProteaseMax Surfactant gave the greatest number of peptide and protein group identifications. This increase was due to better extraction of membrane, nuclear and cytosolic fractions. (4440)

Notes: This paper describes use of the mass-spectrometry-compatible ProteaseMax™ Surfactant to improve protein identification for in-gel digestion applications. The surfactant induced a 1.5−2 fold increase in peptide recovery from gel slices, increased sequence coverage 20−30%, and increased the number of identified proteins. The surfactant also accelerated the digestion process. Maximal in-gel digestion was achieved in as little as one hour, depending on incubation temperature, and peptides were readily recovered from the gel, eliminating the need for postdigestion extraction. (4434)

Notes: These authors analyzed and compared affinity-purified protein complexes from brain homogenates of wild type and huntingtin (Htt) mutant mice by mass spectrometry. Brain tissue from FLAG-tagged wild-type and Htt mice was homogenized in HEPES buffer supplemented with protease inhibitors and RNasin®. After affinity purification, protein complexes were digested using Sequencing-Grade Modified Trypsin and ProteaseMAX™ Trypsin Enhancer prior to mass-spectrometry analysis. (4227)

Notes: The authors of this study used GFP to create a fluorescent reporter that can report the functional state of proteins that are activated by a post-translational event such as enzymatic cleavage or other modification. They created a caspase-activatable GFP (CA-GFP) and analyzed the mechanism of action of the reporter. They subjected purified CA-GFP and GFP to trypsin digestion in the presence of ProteaseMax™ Surfactant, Trypsin Enhancer for matrix-assisted-laser desorption Time-of-Flight (MALDI-TOF) and subsequent MS/MS. They were able to show that the absorbance spectra of CA-GFP does not have the characteristic signature of GFP suggesting that the GFP chromaphore is not mature and in the dark state (Figure 6 in the paper). (4216)

Notes: The authors looked at changes in the rice leaf proteome to understand the molecular mechanisms involved in silicon-induced cadmium tolerance. Total protein was extracted from leaves of plants grown in the presence of sodium silicate or cadmium sulfate. Proteins were separated on 2-D gels, and protein spots were manually excised, reduced and alkylated before digestion with trypsin in the presence of ProteaseMAX™ Surfactant, Trypsin Enhancer. Digests were analyzed via MALDI-TOF MS analysis. The researchers identified 60 proteins, 50 of which were regulated by silicon. (4217)

Notes: The authors of this paper were investigating the role of mycobacterial membrane proteins in pathogenesis of Mycobacterium avium subsp. paratuberculosis, causative agent of Bovine Johne’s disease. Membrane enriched protein fractions, either mucosa-derived membranes (MDM) or culture-derived (CDM) of M. avium paratuberculosis from three cows with clinical disease were examined. Initial 2D DIGE and MALDI-TOF-MS analysis was unsatisfactory, so the researchers subjected the membrane preparations to tube-gel trypsin digestion supplemented with the ProteaseMax™ Surfactant, Trypsin Enhancer. They analyzed the proteins using nanoflow-liquid-chromatography-coupled tandem MS. A total of 130 proteins were detected in both MDM and CDM, with 48 predicted membrane proteins; four were not detected in the CDM, implying differential expression in the host. (4214)

Notes: The authors of this paper investigated the molecular mechanisms through which fumaric acid esters affect the progression and pathology of multiple sclerosis. The authors looked at the activation of the Nrf2 transcriptional pathway which plays a role in defense against oxidative stress. A stable reporter cell line expressing eight copies of the gluthione-S-transferase 2 antioxidative response elements cloned upstream of the luciferase complementary DNA in a pGL4.26 vector. Reporter cells were stimulated with dimethylfumarate for 24 hours and luciferase activity measured using the Luciferase Assay System. A strong dose-dependent induction of antioxidative response elements was observed. Next, the authors looked at the effects of fumaric acid esters on the inhibitor of Nrf2, Keap 1. C-terminal, V5-tagged Keap1 protein was transfected into 293 cells. Forty-eight hours post transfection, cells were treated with dimethyl fumarate. Samples were lysed, Keap1-V5 was immunoprecipitated and gel purified. The protein was digested with trypsin in the presence of ProteaseMAX™ Surfactant, Trypsin Enhancer. Peptide pools were analyzed using liquid chromatography-tandem mass spectrometry. The authors were able to show that the Keap1 protein was modified in response to treatment. (4215)

Notes: The authors of this study investigated the role of lipid raft microdomains that are present in detergent-resistant membranes (DRMs) during Plasmodium ookinete invasion of A. gambiae midguts. The invasion of the mosquito midgut is a critical step in the Plasmodium life cycle and therefore a promising target of transmission-blocking vaccines. The authors analyzed midgut DRMs by tandem MS and also the glycosylphosphatidylinositol-anchored subproteome of A. gambiae midgut brush-border microvilli as well. To prepare the GI-anchored proteins for analysis, an in-gel protein digestion protocol was followed using trypsin in the presence of ProteaseMAX™ Surfactant, Trypsin Enhancer. Nearly 97% of the GI-anchored proteins identified were also present in the DRMs, perhaps providing additional targets for transmission-blocking vaccines. (4218)

Notes: The authors of this paper investigated the mechanism of interaction of HIV-1 Integrase with host cell DNA. To understand the role of the integrase Zn-binding domain, they studied the effect of the Zn ejector 2,2'-dithiobisbenzamide (DIBA) on DNA binding. The covalent modification of integrase by DIBA was evaluated using in-gel tryptic digestion and SELDI mass spectrometry analysis. ProteaseMAX Surfactant was used to enhance protein digestion. (4080)

Notes: These authors sought to identify protein signatures associated with late-stage human colorectal cancer. They used 2D gel electrophoresis to compare the protein signatures of normal tissue and tumor samples. Proteins of interest were excised from gels, trypsin digested and analysed by reverse phase LC-MS. The data were compared to existing databases to try to identify specific proteins or protein interactions associated with late-stage tumor tissue. ProteaseMax Surfactant was used to enhance trypsin digestion. (4084)

Notes: This study describes an enrichment method for cysteinyl adducts of human serum albumin. Electrospray ionization mass spectrometry was used to detect mercaptalbumin and HSA-Cys34 modifications before and after enrichment. After enrichment, mercaptalbumin was no longer observed in mass spectra. Trypsin and ProteaseMax Surfactant were used to prepare samples for mass spectrometry analysis. (4083)

Notes: These authors characterized the venom composition of the water snake Cerberus rynchops. As part of the study, whole venom was trypsinized and fractionated by reverse phase HPLC followed by MALDI-MS/MS analysis. For in-solution tryptic digestion, lyophilized crude venom (500 μg) was dissolved in ammonium bicarbonate and precipitated with ice-cold acetone for 3 h at -15 °C. The pellet was resuspended in 1 ml 50 mM ammonium bicarbonate containing 20 μl of 1% ProteaseMAX Surfactant. After reduction with 10 μl 0.5M DTT (at 56 °C for 20 min) and alkylation with 27 μl of 0.55M iodoacetamide (at room temperature for 15 min in the dark), the venom was digested at 37 °C for 3 h using 18 μg trypsin (1 μg/μl) and 10 μl of 1% ProteaseMAX surfactant to improve tryptic cleavage activity. (4081)

Notes: The microtubule-based cytoskeleton of Toxoplasma gondii is important for cellular invasion. The authors of this paper investigated the tubulin composition and structure
and showed that T. gondii tubulin is extensively altered by post-translational modification. These modifications were analyzed by mass spectrometry. Trypsin digestion of dried gel pieces containing tubulins was performed overnight at 37 °C with the ProteaseMAX Surfactant trypsin enhancer. (4082)

Notes: As part of this study, the authors performed in-gel digestion of expressed protein fragments using trypsin and ProteaseMax Surfactant. The protein of interest was first run on an SDS-PAGE gel and the band excised, cut into 1 x 1mm pieces, reduced with 50 mM DTT and alkylated with 100 mM acrylamide. After destaining with 50 mM 1:1 acetonitrile:ammonium bicarbonate solution, the samples were dried and reconstituted in 12 ng/μL trypsin in the presence of ProteaseMax. The mixture was incubated at 50°C for 1 h before being spun down at 12g for 30 s. The isolated peptides were dried, then reconstituted in 10 μL of 2% acetonitrile/water with 0.1% formic acid before LC-MS/MS analysis. (4079)

Notes: These authors describe a method for comprehensive expression profiling of tissue mitochondria, using swine heart as an example. After protein extraction, a 2-step trypsin digestion method was used to obtain peptides. The authors evaluated use of ProteaseMax as an alternative method to enhance digestion during the first digestion step of the process. The advantages and disadvantages of the method are discussed. (4085)

Notes: These authors used proteolysis and mass spectrometry to identify the specific protein domains involved in copper-dependent phosphorylation. They expressed wildtype and mutant ATP7B in COS1 cells. The band of interest was excised from a gel, cut into small pieces and subjected to tryptic digestion in the presence of ProteaseMax Surfactant. (4086)

Notes: These authors showed that the stomatal initiating factor SPEECHLESSS (SPCH) is a substrate of the kinases MPK3 and MPK6 in vitro, that specific phosphorylation sites on SPCH regulate its activity in vivo, and that components of the stomatal development signaling network modulate SPCH. As part of the study, coomassie-stained gel bands containing the SPCH protein were excised and digested using trypsin and ProteaseMax Surfactant. Mass spectrometry was used to assess phosphorylation at several functionally critical sites. (4087)