Proteomic fingerprinting of apple fruit,juice, and cider via combinatorial peptide ligand libraries and MS analysis

Combinatorial peptide ligand libraries coupled to MS was applied to extensively map the proteome of apple fruit, and to detect its presence in commercial apple juice and cider to evaluate their authenticity and genuineness. Using the Uniprot_Malus database, 96 proteins were detected in apples, among which 30 proteins were specifically captured via combinatorial peptide ligand libraries. Next, three proteins, previously recognized in fruits, were found in apple juice, which were involved in cellular metabolism of fruit maturation and in allergenic reactions. On the other hand, only one Malus allergen was identified in cider beads eluate, demonstrating that the industrial processes did not prevent any negative effects in sensitive subjects. Thus, the present study not only increases the knowledge of the apple proteome but also offers a reliable analytical method to assess quality and genuineness of commercial products, which could be also used to inform consumers about the presence of allergens.     

Sugarcane proteomics: Establishment of a protein extraction method for 2‐DE in stalk tissues and initiation of sugarcane proteome reference map

Sugarcane is an important commercial crop cultivated for its stalks and sugar is a prized commodity essential in human nutrition. Proteomics of sugarcane is in its infancy, especially when dealing with the stalk tissues, where there is no study to date. A systematic proteome analysis of stalk tissue yet remains to be investigated in sugarcane, wherein the stalk tissue is well known for its rigidity, fibrous nature, and the presence of oxidative enzymes, phenolic compounds and extreme levels of carbohydrates, thus making the protein extraction complicated. Here, we evaluated five different protein extraction methods in sugarcane stalk tissues. These methods are as follows: direct extraction using lysis buffer (LB), TCA/acetone precipitation followed by solubilization in LB, LB containing thiourea (LBT), and LBT containing tris, and phenol extraction. Both quantitative and qualitative protein analyses were performed for each method. 2‐DE analysis of extracted total proteins revealed distinct differences in protein patterns among the methods, which might be due to their physicochemical limitations. Based on the 2‐D gel protein profiles, TCA/acetone precipitation‐LBT and phenol extraction methods showed good results. The phenol method showed a shift in pI values of proteins on 2‐D gel, which was mostly overcome by the use of 2‐D cleanup kit after protein extraction. Among all the methods tested, 2‐D cleanup‐phenol method was found to be the most suitable for producing high number of good‐quality spots and reproducibility. In total, 30 and 12 protein spots commonly present in LB, LBT and phenol methods, and LBT method were selected and subjected to eLD‐IT‐TOF‐MS/MS and nESI‐LC‐MS/MS analyses, respectively, and a reference map has been established for sugarcane stalk tissue proteome. A total of 36 nonredundant proteins were identified. This is a very first basic study on sugarcane stalk proteome analysis and will promote the unexplored areas of sugarcane proteome research.     

Identification of avocado (Persea americana) pulp proteins by nano‐LC‐MS/MS via combinatorial peptide ligand libraries

Avocado (Persea americana) proteins have been scarcely studied despite their importance, especially in food related allergies. The proteome of avocado pulp was explored in depth by extracting proteins with capture by combinatorial peptide ligand libraries at pH 7.4 and under conditions mimicking reverse‐phase capture at pH 2.2. The total number of unique gene products identified amounts to 1012 proteins, of which 174 are in common with the control, untreated sample, 190 are present only in the control and 648 represent the new species detected via combinatorial peptide ligand libraries of all combined eluates and likely represent low‐abundance proteins. Among the 1012 proteins, it was possible to identify the already known avocado allergen Pers a 1 and different proteins susceptible to be allergens such as a profilin, a polygalacturonase, a thaumatin‐like protein, a glucanase, and an isoflavone reductase like protein.     

Proteome profile of salt gland‐rich epidermis extracted from a salt‐tolerant tree species

Preparation of proteins from salt‐gland‐rich tissues of mangrove plant is necessary for a systematic study of proteins involved in the plant's unique desalination mechanism. Extraction of high‐quality proteins from the leaves of mangrove tree species, however, is difficult due to the presence of high levels of endogenous phenolic compounds. In our study, preparation of proteins from only a part of the leaf tissues (i.e. salt gland‐rich epidermal layers) was required, rendering extraction even more challenging. By comparing several extraction methods, we developed a reliable procedure for obtaining proteins from salt gland‐rich tissues of the mangrove species Avicennia officinalis. Protein extraction was markedly improved using a phenol‐based extraction method. Greater resolution 1D protein gel profiles could be obtained. More promising proteome profiles could be obtained through 1D‐LC‐MS/MS. The number of proteins detected was twice as much as compared to TUTS extraction method. Focusing on proteins that were solely present in each extraction method, phenol‐based extracts contained nearly ten times more proteins than those in the extracts without using phenol. The approach could thus be applied for downstream high‐throughput proteomic analyses involving LC‐MS/MS or equivalent. The proteomics data presented herein are available via ProteomeXchange with identifier PXD001691.     

Peptide prefractionation is essential for proteomic approaches employing multiple‐reaction monitoring of fruit proteomic research

Off‐gel? IEF has become a popular tool in proteomics research to fractionate peptides or proteins. We conducted a detailed investigation on the fruit proteomics of apple, banana, and strawberry fruit employing Off‐gel? electrophoresis (OGE) as a crucial step to improve the proteome coverage and quantitative proteomic workflows including multiple‐reaction monitoring (MRM). We provide technical details concerning the application of Off‐gel?IEF, nano‐LC–MS detection, and MRM optimization and analysis. Our results demonstrated that the application of OGE is an effective method for peptide fractionation and increased significantly the number of proteins identified by at least ten times, with more total peptides detected and collected. Furthermore, we developed a protocol combining OGE and MRM studies to identify and quantitatively investigate monodehydroascorbate reductase, a key enzyme in the redox and antioxidant system of apple fruit during fruit ripening. Using this method, the quantitative changes in this protein during ripening and in response to ethylene treatment was investigated. Our results provide direct and comprehensive evidence demonstrating the benefits of OGE and its application for both shotgun and quantitative proteomics research.     

应用非同质荷比规则提高蛋白质组学检索结果的可信度和灵敏度

如何筛选合理的数据库匹配结果对于基于质谱的蛋白质组学研究至关重要。但是目前,基于打分体系和反转数据库的筛选方法都无法有效的避免假阳性和假阴性匹配的存在。因此,本文提出了一种系统的搜索策略: 非同质荷比检索规则 (INMZS)。在该策略中,所有匹配结果都需要检查相关匹配质荷比的分享程度,只有那些相关质荷比均为专有匹配时,蛋白质才会被作为可信结果保留,策略还采用迭代搜索方法以提高鉴定低丰度组分的灵敏度。最终,所有的匹配结果由诱饵数据库方法进行评估以得到最终结果列表。INMZS策略在标准蛋白质混合物和大规模人肝蛋白质组数据上进行了模拟及应用,结果显示,INMZS规则和诱饵数据库评估方法的结和可以有效的保证蛋白质组学数据匹配的可信度及灵敏度,可以广泛适用于基于二维凝胶电泳及非shotgun技术的蛋白组学研究中。     

Combination of virtual and experimental 2DE together with ESI LC‐MS/MS gives a clearer view about proteomes of human cells and plasma

Virtual and experimental 2DE coupled with ESI LC‐MS/MS was introduced to obtain better representation of the information about human proteome. The proteins from HEPG2 cells and human blood plasma were run by 2DE. After staining and protein spot identification by MALDI‐TOF MS, the protein maps were generated. The experimental physicochemical parameters (pI/Mw) of the proteoforms further detected by ESI LC‐MS/MS in these spots were obtained. Next, the theoretical pI and Mw of identified proteins were calculated using program Compute pI/Mw ( http://web.expasy.org/compute_pi/pi_tool‐doc.html ). Accordingly, the relationship between theoretical and experimental parameters was analyzed, and the correlation plots were built. Additionally, virtual/experimental information about different protein species/proteoforms from the same genes was extracted. As it was revealed from the plots, the major proteoforms detected in HepG2 cell line have pI/Mw parameters similar to theoretical values. In opposite, the minor protein species have mainly very different from theoretical pI and Mw parameters. A similar situation was observed in plasma in much higher degree. It means that minor protein species are heavily modified in cell and even more in plasma proteome.     

Deciphering key proteins of oil palm (Elaeis guineensis Jacq.) fruit mesocarp development by proteomics and chemometrics

Palm oil is an edible vegetable oil derived from lipid‐rich fleshy mesocarp tissue of oil palm (Elaeis guineensis Jacq.) fruit and is of global economic and nutritional relevance. While the understanding of oil biosynthesis in plants is improving, the fundamentals of oil biosynthesis in oil palm still require further investigations. To gain insight into the systemic mechanisms that govern oil synthesis during oil palm fruit ripening, the proteomics approach combining gel‐based electrophoresis and mass spectrometry was used to profile protein changes and classify the patterns of protein accumulation during these complex physiological processes. Protein profiles from different stages of fruit ripening at 10, 12, 14, 15, 16, 18 and 20 weeks after anthesis (WAA) were analysed by two‐dimensional gel electrophoresis (2DE). The proteome data were then visualised using a multivariate statistical analysis of principal component analysis (PCA) to get an overview of the proteome changes during the development of oil palm mesocarp. A total of 68 differentially expressed protein spots were successfully identified by matrix‐assisted laser desorption/ionisation‐time of flight (MALDI‐TOF/TOF) and functionally classified using ontology analysis. Proteins related to lipid production, energy, secondary metabolites and amino acid metabolism are the most significantly changed proteins during fruit development representing potential candidates for oil yield improvement endeavors. Data are available via ProteomeXchange with identifier PXD009579. This study provides important proteome information for protein regulation during oil palm fruit ripening and oil synthesis.     

Electrospray MS and MALDI imaging show that non‐specific lipid‐transfer proteins (LTPs) in tomato are present as several isoforms and are concentrated in seeds

Non‐specific lipid‐transfer proteins (nsLTPs) are major human allergens in many plant species, albeit their role in plant biochemistry is still undefined. They are found in many plant species, either as one or several isoforms according to the species, and usually they are found to concentrate in the outer part of the fruits. In this work, the characterization of tomato nsLTP isoforms was performed on the three main fractions of Piccadilly tomato fruit (peel, pulp and seeds) by using ultracentrifuge devices with molecular cut‐off able to retain proteins with molecular weight typical of plant LTPs. The isolated proteins were further analysed by LC‐MS, in order to investigate the occurrence and the localization of tomato LTP isoforms. The chromatographic retention times, the molecular masses, the presence of eight cysteine residues in their tertiary structures and the sequence information obtained by MS, although not complete yet, allowed us to identify four different LTP isoforms, not yet reported in the literature, which were found to be concentrated in the seed fractions. None of the molecular masses of these potential LTPs was already present in the UniProtKB/SwissProt database. MALDI imaging experiments confirmed their presence and main localization in seeds, although the actual data hinted at their presence around seeds, rather than exactly in them. These data hint to a complicated scenario concerning LTP proteins in tomato. Copyright © 2014 John Wiley & Sons, Ltd.     

Effects of peptide acetylation and dimethylation on electrospray ionization efficiency

Peptide acetylation and dimethylation have been widely used to derivatize primary amino groups (peptide N‐termini and the ε‐amino group of lysines) for chemical isotope labeling of quantitative proteomics or for affinity tag labeling for selection and enrichment of labeled peptides. However, peptide acetylation results in signal suppression during electrospray ionization (ESI) due to charge neutralization. In contrast, dimethylated peptides show increased ionization efficiency after derivatization, since dimethylation increases hydrophobicity and maintains a positive charge on the peptide under common LC conditions. In this study, we quantitatively compared the ESI efficiencies of acetylated and dimethylated model peptides and tryptic peptides of BSA. Dimethylated peptides showed higher ionization efficiency than acetylated peptides for both model peptides and tryptic BSA peptides. At the proteome level, peptide dimethylation led to better protein identification than peptide acetylation when tryptic peptides of mouse brain lysate were analyzed with LC‐ESI‐MS/MS. These results demonstrate that dimethylation of tryptic peptides enhanced ESI efficiency and provided up to two‐fold improved protein identification sensitivity in comparison with acetylation. Copyright © 2016 John Wiley & Sons, Ltd.     

Identification of putative and potential cross-reactive chickpea (Cicer arietinum) allergens through an in silico approach

BackgroundAllergy has become a key cause of morbidity worldwide. Although many legumes (plants in the Fabaceae family) are healthy foods, they may have a number of allergenic proteins. A number of allergens have been identified and characterized in Fabaceae family, such as soybean and peanut, on the basis of biochemical and molecular biological approaches. However, our understanding of the allergens from chickpea (Cicer arietinum L.), belonging to this family, is very limited.ObjectiveIn this study, we aimed to identify putative and cross-reactive allergens from Chickpea (C. arietinum) by means of in silico analysis of the chickpea protein sequences and allergens sequences from Fabaceae family.MethodsWe retrieved known allergen sequences in Fabaceae family from the IUIS Allergen Nomenclature Database. We performed a protein BLAST (BLASTp) on these sequences to retrieve the similar sequences from chickpea. We further analyzed the retrieved chickpea sequences using a combination of in silico tools, to assess them for their allergenicity potential. Following this, we built structure models using FUGUE: Sequence-structure homology; these models generated by the recognition tool were viewed in Swiss-PDB viewer.ResultsThrough this in silico approach, we identified seven novel putative allergens from chickpea proteome sequences on the basis of similarity of sequence, structure and physicochemical properties with the known reported legume allergens. Four out of seven putative allergens may also show cross reactivity with reported allergens since potential allergens had common sequence and structural features with the reported allergens.ConclusionThe in silico proteomic identification of the allergen proteins in chickpea provides a basis for future research on developing hypoallergenic foods containing chickpea. Such bioinformatics approaches, combined with experimental methodology, will help delineate an efficient and comprehensive approach to assess allergenicity and pave the way for a better understanding of the biological and medical basis of the same.     

Identification and characterization of proanthocyanidins of 16 members of the Rhododendron genus (Ericaceae) by tandem LC–MS

The proanthocyanidins of the leaves of 16 taxa of the Rhododendron genus (Ericaceae) [Rhododendron ‘Catawbiense Grandiflorum’, Rhododendron ‘Cunningham's White’, Rhododendron smirnowii Trautv., Rhododendron calophytum Franch., Rhododendron dichroanthum ssp. scyphocalyx (Balf. f. & Forrest ) Cowan, Rhododendron micranthum Turcz., Rhododendron praevernum Hutch., Rhododendron ungernii Trautv., Rhododendron kaempferi Planch., Rhododendron degronianum ssp. heptamerum var. hondoense ( Nakai ) H. Hara, Rhododendron fortunei Lindl., Rhododendron ponticum L., Rhododendron galactinum Balf. f. ex Tagg., Rhododendron oreotrephes W. W. Sm., Rhododendron brachycarpum ssp. brachycarpum D. Don ex G. Don, and Rhododendron insigne Hemsl. & E. H. Wilson ] were investigated qualitatively by liquid chromatography–mass spectrometry in series. Twenty‐nine dimeric proanthocyanidins based on (epi)catechin and (epi)gallocatechin were detected and characterized on the basis of their unique fragmentation pattern in the negative ion mode tandem mass spectrometry spectra. All of them were extracted for the first time from these sources, and ten of them were not reported previously in nature. The position of the galloyl residue was assigned on the basis of the retro‐Diels–Alder fragmentation and the dehydrated retro‐Diels–Alder fragmentation; it resulted from the loss of gallic acid as a neutral loss in the negative ion mode. Furthermore, four caffeoylquinic acids, six p‐coumaroylquinic acids, epigallocatechin, gallocatechin, catechin, epicatechin, epigallocatechin gallate, catechin gallate, epicatechin gallate, gallocatechin gallate, two quercetin‐O‐hexosides, quercetin‐O‐galloyl‐hexoside, quercetin‐O‐pentoside, quercetin‐O‐rhamnoside, quercetin‐O‐pentoside‐O‐hexoside, quercetin‐O‐rhamnoside‐O‐hexoside, quercetin‐O‐feruloyl‐hexoside, quercetin‐O‐(p‐hydroxy)benzoyl‐hexoside, taxifolin‐O‐pentoside, myricetin‐O‐rhamnoside, two myricetin‐O‐pentosides, three myricetin‐O‐hexosides, and two myricetin‐O‐galloyl‐hexosides were detected and shown to possess characteristic tandem mass spectrometry spectra and were tentatively assigned on the basis of their retention time. Copyright © 2012 John Wiley & Sons, Ltd.     

Combined usage of cascade affinity fractionation and LC‐MS/MS for the proteomics of adult mouse testis

In this report, the proteomics of adult mouse testis were analyzed by the combined usage of cascade affinity fractionation and LC‐MS/MS. The differences between the selected affinity ligands in size, shape, structure, and biochemical characteristics, result in each ligand exhibiting a specific affinity to some protein groups. Therefore, a cascade composition of different ligands can be applied to the fractionation of complex tissue proteins. Ultimately, the fractions collected from cascade affinity fractionation were analyzed by LC‐MS/MS, which resulted in high confidence identification of a total of 1378 non‐redundant mouse testis protein groups, over 2.6 times as many proteins as were detected in the un‐fractionated sample (526). All detected proteins were bioinformatically categorized according to their physicochemical characteristics (such as relative molecular mass, pI, grand average hydrophobicity value, and transmembrane helices), subcellular location, and function annotation. This approach highlighted the sensitivity of this method to a wide variety of protein classes. Utilizing a combination of cascade affinity fractionation and LC‐MS/MS, we have established the largest proteomic database for adult mouse testis at the present time.     

Chlorophyll Breakdown in Senescent Banana Leaves: Catabolism Reprogrammed for Biosynthesis of Persistent Blue Fluorescent Tetrapyrroles

Chlorophyll breakdown is a visual phenomenon of leaf senescence and fruit ripening. It leads to the formation of colorless chlorophyll catabolites, a group of (chlorophyll‐derived bilin‐type) linear tetrapyrroles. Here, analysis and structure elucidation of the chlorophyll breakdown products in leaves of banana (Musa acuminata) is reported. In senescent leaves of this monocot all chlorophyll catabolites identified were hypermodified fluorescent chlorophyll catabolites (hmFCCs). Surprisingly, nonfluorescent chlorophyll catabolites (NCCs) were not found, the often abundant and apparently typical final chlorophyll breakdown products in senescent leaves. As a rule, FCCs exist only fleetingly, and they isomerize rapidly to NCCs in the senescent plant cell. Amazingly, in the leaves of banana plants, persistent hmFCCs were identified that accounted for about 80 % of the chlorophyll broken down, and yellow leaves of M. acuminata display a strong blue luminescence. The structures of eight hmFCCs from banana leaves were analyzed by spectroscopic means. The massive accumulation of the hmFCCs in banana leaves, and their functional group characteristics, indicate a chlorophyll breakdown path, the downstream transformations of which are entirely reprogrammed towards the generation of persistent and blue fluorescent FCCs. As expressed earlier in related studies, the present findings call for attention, as to still elusive biological roles of these linear tetrapyrroles.     

Part II: defining and quantifying individual and co-cultured intracellular proteomes of two thermophilic microorganisms by GeLC-MS2 and spectral counting

The proteome of extremely thermophilic microorganisms affords a glimpse into the dynamics of microbial ecology of high temperature environments. The secretome, or extracellular proteome of these microorganisms, no doubt harbors technologically important enzymes and other thermostable biomolecules that, to date, have been characterized only to a limited extent. In the first of a two-part study on selected thermophiles, defining the secretome requires a sample preparation method that has no negative impact on all downstream experiments. Following efficient secretome purification, GeLC-MS² analysis and prediction servers suggested probable protein secretion to complement experimental data. In an effort to define the extracellular proteome of the extreme thermophilic bacterium Caldicellulosiruptor saccharolyticus, several techniques were considered regarding sample processing to achieve the most in-depth analysis of secreted proteins. Order of operation experiments, all including the C₁₈ bead technique, demonstrated that two levels of sample purification were necessary to effectively desalt the sample and provide sufficient protein identifications. Five sample preparation combinations yielded 71 proteins and the majority described, as enzymatic and putative uncharacterized proteins, anticipate consolidated bioprocessing applications. Nineteen proteins were predicted by Phobius, SignalP, SecretomeP, or TatP for extracellular secretion, and 11 contained transmembrane domain stretches suggested by Phobius and transmembrane hidden Markov model. The sample preparation technique demonstrating the most effective outcome for C. saccharolyticus secreted proteins in this study, involved acetone precipitation followed by the C₁₈ bead method in which 2.4% (63 proteins) of the predicted proteome was identified, including proteins suggested to have secretion and transmembrane moieties.     

Combination of FASP and fully automated 2D‐LC‐MS/MS allows in‐depth proteomic characterization of mouse zymogen granules

Zymogen granule (ZG) constituents play important roles in pancreatic injury and disease. In previous studies, proteomic analyses with rat zymogen granules were separated by two‐dimensional gel electrophoresis or one‐dimensional SDS–PAGE, followed by in‐gel tryptic digestion. In order to overcome the disadvantage of in‐gel digestion and to carry out further in‐depth proteomic analysis of the zymogen granules, in this study, by combining a filter‐aided sample preparation method and fully automated 2D‐LC‐MS/MS technique, 800 ZG proteins were identified with at least two unique peptides for each protein, 75% of which have not been previously reported. The identified proteins revealed broad diversity in protein identity and function. This is the largest dataset of ZG proteome, and also the first dataset of the mouse ZG proteome, which may help elucidate on the molecular architecture of ZGs and their functions. Copyright © 2012 John Wiley & Sons, Ltd.     

A 2D reversed-phase × ion-pair reversed-phase HPLC-MALDI TOF/TOF-MS approach for shotgun proteome analysis

The separation of complex peptide mixtures in shotgun proteome analysis using a 2D separation scheme encompassing reversed-phase × ion-pair reversed-phase (IP-RP) liquid chromatography coupled online to electrospray ion trap mass spectrometry (MS) has been shown earlier to be superior in terms of separation efficiency and technical robustness compared to the classically used separation scheme encompassing strong cation exchange × IP-RP-chromatography in shotgun proteome analysis. In the present study, this novel separation scheme was coupled offline to matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF)/TOF-MS for the analysis of the same sample, a tryptic digest of the cytosolic proteome of the bacterium Corynebacterium glutamicum. Compared to the earlier study, the MALDI-based platform led to a significantly increased number of peptides (7,416 vs. 2,709) and proteins (1,208 vs. 468, without single peptide-based identifications), respectively. This represents the majority of all predicted cytosolic proteins in C. glutamicum. The high proteome coverage, as well as the large number of low-abundant proteins identified with this improved analytical platform, pave the way for new biological studies. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Flavonoid profiling in three citrus varieties native to the Republic of Korea using liquid chromatography coupled with tandem mass spectrometry: contribution to overall antioxidant activity

A mixture of flavonoid components was isolated from the fruit peel of three varieties of citrus native to Republic of Korea, Citrus leiocarpa Hort. ex Tanaka (CLHT), Citrus aurantium L. (CAL) and Citrus erythrosa Hort. (CEH), via 70% methanol extraction followed by ethyl acetate elution over a silica gel cartridge. The flavonoid components of the mixture were analyzed via high‐performance liquid chromatography–tandem mass spectrometry (HPLC‐MS/MS) in positive‐ion mode and a comparison of the reported data. Among 17 characterized components, two flavanones, four flavones and two coumarin derivatives in the fruit peel of the three varieties were identified for the first time. The individual characterized components were quantified via HPLC‐UV. The flavanones dominated in CAL, whereas the flavones prevailed in CLHT and CEH. The antioxidant activity of the flavonoid mixture of the fruit peel was determined via DPPH^?, ABTS^?+ and reducing power assays. The antioxidant activity of CEH and CAL was greater than that of CLHT. Copyright © 2011 John Wiley & Sons, Ltd.     

A Stable Isotopic Pre-digestion Labeling Method for Protein Quantitative Analysis Using Matrix-assisted Laser Desorption/ionization Mass Spectrometry

As an extension of our previous work, here a strategy was demonstrated for protein identification and quantification analyses utilizing a combination of stable isotope chemical labeling with subsequent denaturation, enzymatic digestion and matrix assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). Using [d₀]‐ and [d₆]‐4,6‐dimethoxy‐2‐(methylsulfonyl)pyrimidine ([d₀]‐/[d₆]‐DMMSP), stable isotopic labels were incorporated before digestion. The comparative samples were combined before labeling after digestion, thus biases resulting from differences in sample digestion were avoided and the higher accuracy of quantification could be attained. The labeling was spatial‐selective to particular residues of cysteine, lysine, and tyrosine before denaturation, which could lead to a better universality of the strategy for cysteine‐free proteins. In addition, some lysine residues were blocked after labeling, the partly destroyed recognition sites could simplify the trypsin hydrolysates and hence facilitate the MS complexity. Together, our one‐step labeling strategy combined several desirable properties such as spatial‐selective labeling, reliability of quantitative results, simplification of analysis of complex systems and direct analysis with minimum sample handling. Our results demonstrate the usefulness of the method for analyzing lysozyme in egg white. The method was expected to provide a new powerful tool for comparative proteome research.     

Proteomic profiling of human sera for discovery of potential biomarkers to monitor abstinence from alcohol abuse

Although numerous biomarkers or biomarker candidates have been discovered to detect levels of drinking and intervals of time after last drinking episode, only a few biomarkers have been applied to monitor abstinence in a longer interval (≥6 wks) from alcohol abuse. Considering sample sources, sensitivity, and specificity, new biomarkers from blood with better accuracy are needed. To address this, serum proteomic profiles were compared between pre‐ and post‐ treatment samples from subjects seeking treatment for alcohol abuse and dependence in an intensive 6wk daily outpatient program using high‐abundance plasma protein immunodepletion and LC‐MS/MS techniques. Protein identification, quantification, candidate biomarker selection, and prioritization analyses were carried out. Among the 246 quantified serum proteins, abundance of 13 and 45 proteins in female and male subjects were significantly changed (p ≤ 0.05), respectively. Of these biomarker candidate proteins, 2 (female) and 8 (male) proteins were listed in category 1, with high area under the receiver operating characteristic curve, sensitivity, specificity, and fold change. In summary, several new biomarker candidates have been identified to monitor abstinence from alcohol abuse.