Nano LC-MS Research Articles

Preparation of polyoxometalate modified magnetic metal–organic framework and application to the enrichment of phosphopeptides

Herein, polyoxometalate modified magnetic metal–organic framework (Fe3O4@PDA@MOF-POM) was successfully prepared and then applied to the enrichment of phosphopeptides. Fe3O4@PDA@MOF-POM can bind with phosphopeptides through ZrO clusters provided by the MOF layer and metal oxide originated from Zr-W-POM. The magnetic nanomaterial had high sensitivity (0.1 fmol), good reusability (10 cycles), and high selectivity (α-casein: β-casein: bovine serum albumin = 1: 1: 5000). Coupling with MALDI-TOF MS determination, non-fat milk was selected to preliminarily explore the applicability of Fe3O4@PDA@MOF-POM. Furthermore, 241 phosphopeptides derived from 232 phosphoproteins and 99 phosphopeptides attributed to 89 phosphoproteins were identified in human serum and saliva by nano LC-MS. This work paves a new way for the recognition of phosphopeptides in bio-samples.

Fungal and bacterial species richness in biodeteriorated seventeenth century Venetian manuscripts

Historical paper documents are susceptible to complex degradation processes, including biodeterioration, which can progressively compromise their aesthetic and structural integrity. This study analyses seventeenth century handwritten historical letters stored at the Correr Museum Library in Venice, Italy, exhibiting pronounced signs of biodeterioration. The techniques used encompassed traditional colony isolation on agar plates and proteomics analyses, employing nanoscale liquid chromatography coupled with high-resolution mass spectrometry (nano-LC–MS). Fluorescence microscopy was used for the first time in the historical paper biodeterioration context to supplement the conventional stereoscopic, optical, and scanning electron microscopic imaging techniques. This method enables the visualisation of microorganisms beyond and beneath the paper’s surface through their natural intrinsic autofluorescence in a non-invasive and non-destructive way. The results demonstrate a diverse, complex, and abundant microbiota composed of coexisting fungal and bacterial species (Ascomycota, Mucoromycota, Basidiomycota, Proteobacteria, and Actinobacteria), along with mite carcasses, insects, parasites, and possibly protists. Furthermore, this study reveals certain species that were not previously documented in the biodeterioration of historical paper, including human pathogens, such as Histoplasma capsulatum, Brucella, Candida albicans, and species of Aspergillus (A. flavus, A. fumigatus, A. oryzae, A. terreus, A. niger) known to cause infections or produce mycotoxins, posing substantial risk to both artefacts and humans.

Preparation of high-efficiency titanium ion immobilized magnetic graphite nitride nanocomposite for phosphopeptide enrichment

BackgroundProtein phosphorylation has been implicated in life processes including molecular interaction, protein structure transformation, and malignant disease. An in-depth study of protein phosphorylation may provide vital information for the discovery of early biomarkers. Mass spectrometry (MS)-based techniques have become an important method for phosphopeptide identification. Nevertheless, direct detection remains challenging because of the low ionization efficiency of phosphopeptides and serious interference from non-phosphopeptides. There is a great need for an efficient enrichment strategy to analyze protein phosphorylation prior to MS analysis. ResultsIn this study, a novel nanocomposite was prepared by introducing titanium ions into two-dimensional magnetic graphite nitride. The nanocomposite was combined with immobilized metal ion affinity chromatography (IMAC) and anion-exchange chromatography mechanisms for phosphoproteome research. The nanocomposite had the advantages of a large specific surface (412.9 m2 g−1), positive electricity (175.44 mV), and excellent magnetic property (35.7 emu g−1). Moreover, it presented satisfactory selectivity (α-casein:β-casein:bovine serum albumin = 1:1:5000), a low detection limit (0.02 fmol), great recyclability (10 cycles), and high recovery (92.8%). The nanocomposite demonstrated great practicability for phosphopeptides from non-fat milk, human serum, and saliva. Further, the nanocomposite was applied to enrich phosphopeptides from a more complicated specimen, A549 cell lysate. A total of 890 phosphopeptides mapping to 564 phosphoproteins were successfully detected with nano LC-MS. SignificanceWe successfully designed and developed an efficient analysis platform for phosphopeptides, which includes protein digestion, phosphopeptide enrichment, and MS detection. The MS-based enrichment platform was further used to analyze phosphopeptides from complicated bio-samples. This work paves the way for the design and preparation of graphite nitride-based IMAC materials for phosphoproteome analysis.

Cerium ions immobilized magnetic graphite nitride decorated with L-Alanyl-L-Glutamine as new chelator for enrichment of phosphopeptides.

Cerium ions immobilized magnetic graphite nitride materialhave been prepared using L-Alanyl-L-Glutamine as the new chelator. The resulting Fe3O4/g-C3N4-L-Ala-L-Gln-Ce4+, as an immobilized metal ion affinity chromatography (IMAC) sorbent, was reusable. This is due to the strong coordination interaction between L-Alanyl-L-Glutamine and cerium ions. After a series of characterizations, the magnetic nanocomposite showed high surface area, good hydrophilicity, positive electricity, and magnetic response. Fe3O4/g-C3N4-L-Ala-L-Gln-Ce4+ had high sensitivity (0.1 fmol), selectivity (α-/β-casein/bovine serum albumin, 1:1:5000), and good recyclability (10 cycles). A total of 647 unique phosphopeptides mapped to 491 phosphoproteins were identified from A549 cell lysate by nano LC-MS analysis.

Mass Spectrometry based identification of site-specific proteomic alterations and potential pathways underlying the pathophysiology of schizophrenia.

Schizophrenia (SZ) is a complex multifactorial disorder that affects 1% of the population worldwide with no available effective treatment. Although proteomic alterations are reported in SZ however proteomic expression aberrations among different brain regions are not fully determined. Therefore, the present study aimed spatial differential protein expression profiling of three distinct regions of SZ brain and identification of associated affected biological pathways in SZ progression. Comparative protein expression profiling of three distinct autopsied human brain regions (i.e., substantia nigra, hippocampus and prefrontal cortex) of SZ was performed with respective healthy controls. Using two-dimensional electrophoresis (2DE)-based nano liquid chromatography tandem mass spectrometry (Nano-LC MS /MS) analysis, 1443 proteins were identified out of which 58 connote to be significantly dysregulated, representing 26 of substantia nigra,14 of hippocampus and 18 of prefrontal cortex. The 58 differentially expressed proteins were further analyzed using Ingenuity pathway analysis (IPA). The IPA analysis provided protein-protein interaction networks of several proteins including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kb), extracellular signal regulated kinases 1/2 (ERK1/2), alpha serine / Threonine-protein kinase (AKT1), cellular tumor antigen p53 (TP53) and amyloid precursor protein (APP), holding prime positions in networks and interacts with most of the identified proteins and their closely interacting partners. These findings provide conceptual insights of novel SZ related pathways and the cross talk of co and contra regulated proteins. This spatial proteomic analysis will further broaden the conceptual framework for schizophrenia research in future.

Mouse milk immunoglobulin G Fc-linked N-glycosylation nano-LC–MS analysis in a model of vancomycin exposure during pregnancy

Breast milk immunoglobulin G (IgG) plays an important role in the transfer of passive immunity in early life and in shaping the neonatal immune system through N-glycan-mediated effector functions. Currently, there are no protocols available to analyze breast milk IgG-Fc glycosylation in mouse models. Therefore, we developed and validated a glycoproteomic workflow for the medium-throughput subclass-specific nano-LC-MS analysis of IgG enriched from small milk volumes of lactating mice. With the established methods, the IgG glycopatterns in a mouse model of antibiotic use during pregnancy and increased asthma susceptibility in the offspring were analyzed. Pregnant BALB/c mice were treated with vancomycin during gestation days 8-17 and IgG1F, IgG2, and IgG3-Fc glycosylation was subsequently analyzed in maternal serum, maternal breast milk, and offspring serum on postnatal day 15. The IgG glycosylation profiles of mouse maternal milk and serum revealed no significant differences within the glycoforms quantified across subclasses. However, vancomycin use during pregnancy was associated with changes in IgG-Fc glycosylationin offspring serum, shown by the decreased relative abundance of the IgG1F-G1 and IgG3-G0 glycoforms, together with the increased relative abundance of the IgG3-G2 and S1 glycoforms. The workflow presented will aid in the emerging integrative multi-omics- and glycomics-oriented milk analyses both in rodent models and human cohorts for a better understanding of mother-infant immunological interactions.

Differential proteomic profile of X- and Y- sorted Sahiwal bull semen

The identification of differential proteins between X- and Y-sperm may be useful for immunological sexing of sperm. Hence, the present study was aimed to compare the protein profile of X- and Y-sorted Sahiwal bull semen using SDS-PAGE and Liquid Chromatography coupled with Mass Spectrometry (Nano LC-MS). Semen sample (n = 6) were categorized into three groups i.e., group I (X-sorted), group II (Y-sorted) and control group (both X- and Y- sperms). SDS PAGE revealed specific proteins of molecular weight between 18 and 24 kDa and between 30 and 37 kDa were present in X-sorted sperms. Also, band corresponding to 25 kDa was specific to Y-sorted sperms. Data obtained from Nano LC/MS is analysed by search engine database i.e., MASCOT and SEQUEST HT. Total, 241 proteins were identified, out of which 113 were differentially expressed between X- and Y-sorted sperms, in which 54 proteins showed at least two unique peptides. Out of 54 proteins, 27 were upregulated in X-sorted sample, 3 were upregulated in Y-sorted sample and 24 were differentially downregulated. Highly upregulated protein in X-sperm viz. Armadillo repeat containing 12 protein, NDC1 transmembrane nucleoporin, β-nerve growth factor, C-type natriuretic peptide, Nucleobindin-2, Phosphoglycerate mutase 2, Calmodulin along with one uncharacterised protein having accession number F1MN9 may have potential to be used as biomarker for separating X and Y sperm.

Differential expression of Lumican, Mimecan, Annexin A5 and Serotransferrin in ectopic and matched eutopic endometrium in ovarian endometriosis: a case-control study

Aim Endometriosis is a debilitating disease marked by recurrent gynecological proliferations. The present study aimed at performing differential proteomic analysis of matched eutopic and ectopic endometrium from women with ovarian endometriosis. Materials and methods Proteomes were resolved using nano LC–MS and further identified and quantified using ProteinLynx Global SERVER (PLGS) software. Selected proteins were further chosen for validation by real time-polymerase chain reaction (RT-PCR). Results The protein profiles uncovered several differentially expressed proteins in the diseased sample (ectopic endometrium) as compared to the reference sample (eutopic endometrium). The study involved an advanced proteomic approach, nano LC–MS, and validates for the first time the upregulation of Mimecan and Lumican proteins in endometriosis. Conclusions These proteins may hence prove as potentially useful tools in the search for diagnostic markers for early detection of the disease.

Taenia solium proteins: a beautiful kaleidoscope of pro and anti-inflammatory antigens

ABSTRACT Background: Neurocysticercosis (NCC) is an acquired infection of central nervous system associated with epileptic seizures. The parasite ‘Taenia solium’ causes this disease and has a complex life cycle and molts into various stages that influence the host-parasite interaction. The disease has a long asymptomatic phase with viable cyst and degeneration of cyst and leaking cyst fluid has been associated with symptomatic phase. The parasite proteome holds the answers and clues to this complex clinical presentation and hence unraveling of proteome of parasite antigens is needed for better understanding of host-parasite interactions. Objective: To understand the proteome make-up of T. solium cyst vesicular fluid (VF) and excretory secretory proteins (ESPs). Methodology: The VF and ESPs for the study were prepared from cyst harvested from naturally infected swine. The samples were prepared for nano LC-MS by in-tube digestion of proteins. The spectra obtained were annotated and enrichment analysis was performed and in silico analysis was done. Results: T. solium VF and ESPs have 206 and 247 proteins of varied make-up including pro-inflammatory and anti-inflammatory nature. Conclusions: Due to varied make-up of VF and ESPs it can generate complex humoral and cellular immune response.

Preparation of Novel Zwitterionic Monolith for Capillary Electrochromatography and Nano LC–MS Applications

Herein, a zwitterionic organic polymer monolithic stationary phase was prepared based on the in situ thermal-initiated copolymerizing 3-dimethyl-(3-(N-methacrylamido) propyl) ammonium propane sulfonate (DMMPPS) and pentaerythritol triacrylate (PETA) in a binary porogenic solvent consisting of MeOH and H2O. An HILIC/RP dual separation mechanism was observed on this optimised poly(DMMPPS-co-PETA) monolithic column and the composition of the mobile phase corresponding to the transition from the HILIC to the RP mode was around 30% ACN in water. The proposed monolithic column was successfully applied to separate 12 polar neurotransmitters in a nano LC–MS mode. About ~ 27 μm plate height (corresponding to column efficiency of ~ 93,000 plates/m) was obtained at the linear velocity of 1 mm/s. Meantime, the DMMPPS-based monolith exhibited good mechanical stability and excellent separation performance for nucleic acid bases, nucleosides and nucleotides for capillary electrochromatography (CEC). Relative standard deviations (RSD%) of the retention times for five nucleic acid bases and nucleosides were in the range of 0.15–0.42% (run-to-run, n = 3) and 2.63–4.50% (column-to-column, n = 3), respectively. Our work demonstrated that the zwitterionic monolith columns could be an effective separation tool for analysis of hydrophilic substances both on the HI-CEC and nano LC–MS mode.

Measurement of ultra-trace level of intact oxytocin in plasma using SALLE combined with nano-LC–MS

Measurement of peptides such as oxytocin in plasma is a critical challenge in clinical research because of their extreme low concentrations as well as the tremendous interferencing substances co-presented in plasma. In this study, we developed an efficient salt-out assisted liquid-liquid extraction (SALLE) to treat plasma, and then analyzed the samples using nano-LC–MS to quantify intact oxytocin (OT) in human and rat plasmas. Our results showed that the use of SALLE (Isopropanol/K2HPO4 (4 M)) allows efficient removal of various disrupters, including proteins, inorganic salts, and lipids, which helps avoid the risk of blocked capillary columns and matrix effects. Moreover, instant SALLE can reduce the possible binding between OT and proteins, thus allowing high repeatability of OT extraction from the original plasma. This combination of SALLE and nano-LC–MS method provided in the end a 1 pg/m L of detection limit. Comparative analysis showed that the concentration of OT in the plasma taken from 12 volunteers ranged from 3 to 214 pg/m L, about one order less than those in the plasma of rats. Compared to the previously reported LC–MS and immunoassay methods, the combination of SALLE and nano-LC–MS permits reliable measurement of intact OT even in human plasma. Our approach may be an alternative method for quantitative determination of other ultra-trace peptides in plasma, which would help the investigators understand the role of peptides in behaviours and diseases.

Ultracentrifugation versus kit exosome isolation: nanoLC-MS and other tools reveal similar performance biomarkers, but also contaminations.

Aim:For isolation of exosomes, differential ultracentrifugation and an isolation kit from a major vendor were compared.Materials & methods:‘Case study’ exosomes isolated from patient-derived cells from glioblastoma multiforme and a breast cancer cell line were analyzed.Results:Transmission electron microscopy, dynamic light scattering, western blotting, and so forth, revealed comparable performance. Potential protein biomarkers for both diseases were also identified in the isolates using nanoLC–MS. Western blotting and nanoLC–MS also revealed negative exosome markers regarding both isolation approaches.Conclusion:The two isolation methods had an overall similar performance, but we hesitate to use the term ‘exosome isolation’ as impurities may be present with both isolation methods. NanoLC–MS can detect disease biomarkers in exosomes and is useful for critical assessment of exosome enrichment procedures.

Proteome Profiling of 1 to 5 Spiked Circulating Tumor Cells Isolated from Whole Blood Using Immunodensity Enrichment, Laser Capture Microdissection, Nanodroplet Sample Processing, and Ultrasensitive nanoLC-MS.

Proteome profiling of circulating tumor cells (CTCs) can provide crucial insight into disease progression and the role of CTCs in tumor metastasis. We describe an integrated workflow to measure global protein expression in 1-5 spiked CTCs enriched from whole blood by immunodensity gradient centrifugation. Enriched CTCs were purified and collected by laser capture microdissection, prepared using a recently developed nanodroplet-based processing platform (nanoPOTS), and finally analyzed by ultrasensitive nanoLC-MS/MS. The workflow was capable of identifying an average of 164 and 607 protein groups from samples comprising 1 and 5 LNCaP cells, respectively, that were isolated from human whole blood. A panel of prostate cancer-specific proteins were identified and quantified, which was used to differentiate between spiked CTCs and white blood cells.

Comparative proteomic analysis of cat eye syndrome critical region protein 1- function in tumor-associated macrophages and immune response regulation of glial tumors

IntroductionTumor associated macrophages (TAMs) promote tumor development, angiogenesis and distal metastasis. In previous studies, we showed that Cat Eye Syndrome Critical Region Protein 1 (CECR1) is expressed by M2-like TAMs in human glioma samples. CECR1 promoted M2 TAMs differentiation and affected glioma cell proliferation and migration. Here we investigated the proteomic profile of TAMs expressing CECR1 in absence or presence of glioma cells.ResultsCECR1 siRNA transfection upregulated 67 proteins in THP-1-derived Macrophages (MQs). Pathway annotation mapped this set to 3 major pathways relevant for MQ function, including ‘MHC-I antigen presentation’, ‘phagosome maturation’ and ‘endocytosis’. Co-culture of siCECR1 THP-1-derived MQs with U87 glioma cells attenuated the changes observed on protein and mRNA level in response to MQ CECR1 silencing. SiCECR1 in U87 co-cultured MQs was associated with an IL-10low, IL-12high M1-like phenotype. In U87 co-culture conditions, SiCECR1 also downregulated S20 proteasome complex proteins PSMA5, PSMA7, PSMC6 and PSMD8. This protein profile was linked to a low proliferation rate of siCECR1 MQs. Overlap analysis identified S100A9 and PLAU as CECR1-related proteins that were significantly correlated with expression of CECR1 and macrophage lineage markers in three large public GBM datasets.ConclusionThis study reports the molecular pathways and key molecules that are mediated by CECR1 function in THP- 1-derived MQs and TAMs in glioma.MethodsPMA-treated THP-1 cells (MQs) were siRNA transfected for CECR1 in vitro, with or without stimulation of the primary glioma cell line U87. Lysates were analyzed by (nano)LC-MS. Significant altered protein levels were identified (P < 0.05), followed by pathway annotation.

Serum ApoA4 levels predicted the progression of renal impairment in T2DM.

Among multiple causes, diabetic nephropathy (DN) is the major underlying renal disease that leads to end-stage renal disease (ESRD), and early diagnosis can effectively prevent or delay the progression to ESRD. Therefore, the current study aimed to develop noninvasive, accurate detection markers. For this study, 62 diabetes mellitus (DM) patients, 59 DN patients and 21 healthy controls (HCs) were recruited. All participants' serum samples were subjected to concavanalin (Con) A affinity chromatography, which utilizes glycoproteins to discover potential markers. From nano LC-MS and Western blot analysis, apolipoprotein A-IV (ApoA4) was selected which featured a gradual, almost twofold increase in the order of HC, DM and DN. In the Con A-based ELISA, the DM group was 1.91-fold higher than the HC group, while the DN group was 2.56-fold higher than the HCs and 1.33-fold higher than the DM group. In addition, significant positive correlations were observed between ApoA4 and blood urea nitrogen levels and between ApoA4 and creatine levels, while significant negative correlations were seen between serum protein levels and between serum albumin levels in comparisons of DM and DN samples. Serum Con A-bound ApoA4 levels were higher in the DM group than in HCs, and further increased in the DN group. Levels of ApoA4 were positively correlated with blood urea nitrogen and creatine, but negatively correlated with serum protein and albumin. This evidence supports serum Con A-bound ApoA4 as a circulating marker for predicting the progression of renal impairment in DM patients.

Automated SPE and nanoLC–MS analysis of somatostatin

ABSTRACTPlasma peptides are widely used in clinical diagnosis and therapy monitoring. Our aim was an efficient system development for the enrichment of small, low abundant peptides from plasma by robotized sample preparation. The automation of SPE yields additional time saving and improves system robustness and repeatability. Automation is based on combining a Waters SPE kit with Oasis HLB sorbent and a multichannel liquid handling workstation with cheap commercially available electronic devices such as a programmable logic controller (PLC) and an AVR controller. Reversed phase nano liquid chromatography coupled with a sensitive quadrupole time of flight mass spectrometer (QTOF MS) was used for the quantitative determination of somatostatin (SST). We quantified SST from mouse plasma, where lower limit of detection (LLOD) and lower limit of quantification (LLOQ) were 2.5 and 8.3 fmol/ml, respectively. We investigated linearity of response, accuracy, precision, recovery, reproducibility and stability of SST during both short-term sample processing and long-term storage. This method allows reliable quantification of plasma peptides. The developed automated sample preparation solid phase extraction method can be easily and conveniently adopted for different volumes and amounts of sample in routine analysis using controlled vacuum. The highest advantage is enhanced reproducibility, which makes it suitable for the investigations of large sample cohorts in clinical studies.

Nano LC-MS Based Proteomic Analysis as a Predicting Approach to Study Cellular Responses of Carbon Nanotubes.

Nano-bio interface has been paid much attention recently, though with the lack of methodology to predict the potential responses in biological systems such as cells induced by nanomaterials. In this study, we described a proteomic approach to investigate the proteome change in K562 cells exposed to oxidized single-walled carbon nanotubes (o-SWCNTs). 605 proteins were identified by semi-quantitative proteomic analysis (SQPA), including 29 significantly changed proteins with spectra count (SpC) ratios lager than 2 or less than 0.5. Three of them including HBA, CFL1 and LMAN2 were further validated by western blotting. The differential proteins were further classified by Ingenuity Pathways Analysis (IPA) to integrate them into a signaling network. Based on the information by this network, we predict that o-SWCNT treatment activated cell aggregation, decreased cell migration, but had no effect on cell death. And these cellular responses were further experimentally demonstrated. The protein signaling network established in this study would greatly benefit the studies on the bio-applications of o-SWCNTs and their toxicity studies. Our study demonstrated that proteomics could be used as a predicting tool to study nano-bio interface at cellular level.

Proteins involved in biophoton emission and flooding-stress responses in soybean under light and dark conditions.

To know the molecular systems basically flooding conditions in soybean, biophoton emission measurements and proteomic analyses were carried out for flooding-stressed roots under light and dark conditions. Photon emission was analyzed using a photon counter. Gel-free quantitative proteomics were performed to identify significant changes proteins using the nano LC-MS along with SIEVE software. Biophoton emissions were significantly increased in both light and dark conditions after flooding stress, but gradually decreased with continued flooding exposure compared to the control plants. Among the 120 significantly identified proteins in the roots of soybean plants, 73 and 19 proteins were decreased and increased in the light condition, respectively, and 4 and 24 proteins were increased and decreased, respectively, in the dark condition. The proteins were mainly functionally grouped into cell organization, protein degradation/synthesis, and glycolysis. The highly abundant lactate/malate dehydrogenase proteins were decreased in flooding-stressed roots exposed to light, whereas the lysine ketoglutarate reductase/saccharopine dehydrogenase bifunctional enzyme was increased in both light and dark conditions. Notably, however, specific enzyme assays revealed that the activities of these enzymes and biophoton emission were sharply increased after 3 days of flooding stress. This finding suggests that the source of biophoton emission in roots might involve the chemical excitation of electron or proton through enzymatic or non-enzymatic oxidation and reduction reactions. Moreover, the lysine ketoglutarate reductase/saccharopine dehydrogenase bifunctional enzyme may play important roles in responses in flooding stress of soybean under the light condition and as a contributing factor to biophoton emission.

A novel chemical footprinting approach identifies critical lysine residues involved in the binding of receptor-associated protein to cluster II of LDL receptor-related protein.

Tandem mass tags (TMTs) were utilized in a novel chemical footprinting approach to identify lysine residues that mediate the interaction of receptor-associated protein (RAP) with cluster II of LDL (low-density lipoprotein) receptor (LDLR)-related protein (LRP). The isolated RAP D3 domain was modified with TMT-126 and the D3 domain-cluster II complex with TMT-127. Nano-LC-MS analysis revealed reduced modification with TMT-127 of peptides including Lys(256), Lys(270) and Lys(305)-Lys(306) suggesting that these residues contribute to cluster II binding. This agrees with previous findings that Lys(256) and Lys(270) are critical for binding cluster II sub-domains [Fisher, Beglova and Blacklow (2006) Mol. Cell 22, 277-283]. Cluster II-binding studies utilizing D3 domain variants K(256)A, K(305)A and K(306)A now showed that Lys(306) contributes to cluster II binding as well. For full-length RAP, we observed that peptides including Lys(60), Lys(191), Lys(256), Lys(270) and Lys(305)-Lys(306) exhibited reduced modification with TMT in the RAP-cluster II complex. Notably, Lys(60) has previously been implicated to mediate D1 domain interaction with cluster II. Our results suggest that also Lys(191) of the D2 domain contributes to cluster II binding. Binding studies employing the RAP variants K(191)A, K(256)A, K(305)A and K(306)A, however, revealed a modest reduction in cluster II binding for the K(256)A variant only. This suggests that the other lysine residues can compensate for the absence of a single lysine residue for effective complex assembly. Collectively, novel insight has been obtained into the contribution of lysine residues of RAP to cluster II binding. In addition, we propose that TMTs can be utilized to identify lysine residues critical for protein complex formation.

Proteome analysis during pod, zygotic and somatic embryo maturation of Theobroma cacao

Two dimensional electrophoresis and nano-LC–MS were performed in order to identify alterations in protein abundance that correlate with maturation of cacao zygotic and somatic embryos. The cacao pod proteome was also characterized during development. The recently published cacao genome sequence was used to create a predicted proteolytic fragment database. Several hundred protein spots were resolved on each tissue analysis, of which 72 variable spots were subjected to MS analysis, resulting in 49 identifications. The identified proteins represent an array of functional categories, including seed storage, stress response, photosynthesis and translation factors. The seed storage protein was strongly accumulated in cacao zygotic embryos compared to their somatic counterpart. However, sucrose treatment (60gL−1) allows up-regulation of storage protein in SE. A high similarity in the profiles of acidic proteins was observed in mature zygotic and somatic embryos. Differential expression in both tissues was observed in proteins having high pI. Several proteins were detected exclusively in fruit tissues, including a chitinase and a 14-3-3 protein. We also identified a novel cacao protein related to known mabinlin type sweet storage proteins. Moreover, the specific presence of thaumatin-like protein, another sweet protein, was also detected in fruit tissue. We discuss our observed correlations between protein expression profiles, developmental stage and stress responses.