Proteomics analysis of human breast milk to assess breast cancer risk

Detection of breast cancer (BC) in young women is challenging because mammography, the most common tool for detecting BC, is not effective on the dense breast tissue characteristic of young women. In addition to the limited means for detecting their BC, young women face a transient increased risk of pregnancy‐associated BC. As a consequence, reproductively active women could benefit significantly from a tool that provides them with accurate risk assessment and early detection of BC. One potential method for detection of BC is biochemical monitoring of proteins and other molecules in bodily fluids such as serum, nipple aspirate, ductal lavage, tear, urine, saliva and breast milk. Of all these fluids, only breast milk provides access to a large volume of breast tissue, in the form of exfoliated epithelial cells, and to the local breast environment, in the form of molecules in the milk. Thus, analysis of breast milk is a non‐invasive method with significant potential for assessing BC risk. Here we analyzed human breast milk by mass spectrometry (MS)‐based proteomics to build a biomarker signature for early detection of BC. Ten milk samples from eight women provided five paired‐groups (cancer versus control) for analysis of dysregulatedproteins: two within woman comparisons (milk from a diseased breast versus a healthy breast of the same woman) and three across women comparisons (milk from a woman with cancer versus a woman without cancer). Despite a wide range in the time between milk donation and cancer diagnosis (cancer diagnosis occurred from 1 month before to 24 months after milk donation), the levels of some proteins differed significantly between cancer and control in several of the five comparison groups. These pilot data are supportive of the idea that molecular analysis of breast milk will identify proteins informative for early detection and accurate assessment of BC risk, and warrant further research. Data are available via ProteomeXchange with identifier PXD007066.     

Proteomics analysis of human breast milk by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) coupled with mass spectrometry to assess breast cancer risk

Breast cancer (BC) is one of the most common cancers and one of the most common causes for cancer-related mortality. Discovery of protein biomarkers associated with cancer is considered important for early diagnosis and prediction of the cancer risk. Protein biomarkers could be investigated by large-scale protein investigation or proteomics, using mass spectrometry (MS)-based techniques. Our group applies MS-based proteomics to study the protein pattern in human breast milk from women with BC and controls and investigates the alterations and dysregulations of breast milk proteins in comparison pairs of BC versus control. These dysregulated proteins might be considered potential future biomarkers of BC. Identification of potential biomarkers in breast milk may benefit young women without BC, but who could collect the milk for future assessment of BC risk. Previously we identified several dysregulated proteins in different sets of human breast milk samples from BC patients and controls using gel-based protein separation coupled with MS. Here, we performed 2D-PAGE coupled with nano-liquid chromatography–tandem MS (nanoLC-MS/MS) in a small-scale study on a set of six human breast milk pairs (three BC samples vs. three controls) and we identified several dysregulated proteins that have potential roles in cancer progression and might be considered potential BC biomarkers in the future.     

Comparative analysis of prostate‐specific antigen by two‐dimensional gel electrophoresis and capillary electrophoresis

Serum levels of Prostate‐Specific Antigen (PSA) are not fully specific for prostate cancer (PCa) diagnosis and several efforts are focused on searching to improve PCa markers through the study of PSA subforms that could be cancer associated. We have previously reported by 2DE a decrease in the sialic acid content of PSA from PCa compared to benign prostatic hyperplasia patients based on the different proportion of the PSA spots. However, faster and more quantitative techniques, easier to automate than 2DE, are desirable. In this study, we examined the potential of CE for resolving PSA subforms in different samples and compared the results with those obtained by 2DE. We first fractionated by OFFGEL the subforms of PSA from seminal plasma according to their pI s and analyzed each separated fraction by 2DE and CE. We also analyzed PSA and high pI PSA, both from seminal plasma, and PSA from urine of a PCa patient. These samples with different PSA spots proportions by 2DE, due to different posttranslational modifications, also presented different CE profiles. This study shows that CE is a useful and complementary technique to 2DE for analyzing samples with different PSA subforms, which is of high clinical interest.     

Sample pooling in 2-D gel electrophoresis: a new approach to reduce nonspecific expression background

Protein expression alterations unrelated to an investigated phenotype are accumulated in most cell line models during establishment. Performing a whole proteome screening of lymphoma cell lines, we established a method to reduce the influence of protein expression unrelated to the distinct investigated phenotype. In 2-D PAGE, the comprehensive analysis of a large number of protein spots would be simplified by pooling cell line samples of the investigated phenotype. Applying this pooling approach, unrelated alterations of single samples are 'muted' by dilution. Analysing two different lymphoma subtypes (follicular and mantle cell lymphoma) by this method, spots originating in only single cell lines were reduced by 72% (650/900), whereas even modestly altered expression of protein spots detected in all lines were reliably detected in the pooled protein gels. We conclude that our pooling approach is a preferable approach to reliably detect a common protein expression pattern and may even allow proteomic analysis of clinical samples with limited amounts of sample material, even with minimal cell numbers as low as 1 x 10(6).     

Development of a rapid and improved two‐dimensional electrophoresis method for the separation of protein lysate

Researchers frequently use two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) prior to mass spectrometric analysis in a proteomics approach. The i2D‐PAGE method, which ‘inverts’ the dimension of protein separation of the conventional 2D‐PAGE, is presented in this publication. Protein lysate of Channa striata, a freshwater snakehead fish, was separated based on its molecular weight in the first dimension and its isoelectric point in the second dimension. The first‐dimension separation was conducted on a gel‐free separation device, and the protein mixture was fractionated into 12 fractions in chronological order of increasing molecular weight. The second‐dimension separation featured isoelectric focusing, which further separated the proteins within the same fraction according to their respective isoelectric point. Advantages of i2D‐PAGE include better visualisation of the isolated protein, easy identification on protein isoforms, shorter running time, customisability and reproducibility. Erythropoietin standard was applied to i2D‐PAGE to show its effectiveness for separating protein isoforms. Various staining methods such as Coomassie blue staining and silver staining are also applicable to i2D‐PAGE. Overall, the i2D‐PAGE separation method effectively separates protein lysate and is suitable for application in proteomics research.     

A competent extraction method of plant proteins for 2-D gel electrophoresis

The efficient extraction of high‐quality proteins is a key factor for a successful proteomic analysis approach. In the method suggested here, absolute ethanol containing 10 mM DTT was used to precipitate the proteins in plant tissue homogenates followed by their resuspension in a urea‐/thiourea‐ and NP‐40‐containing solution. Protein profiles were examined on pH 3–11 non‐linear IEF strips and SDS‐PAGE and compared with extracts using the established method of acetone‐10% TCA/0.07% 2‐mercaptoethanol precipitation (V. Méchin et al., Methods Mol. Biol. 2006, 355, 1–8). In addition to protein profile similarity for the two extracts, the acidic part of the acetone containing 10% TCA/0.07% 2‐mercaptoethanol extraction showed protein spots with high molecular weight in the range of 250–150 kDa, while the ethanol containing 10 mM DTT extracts indicated extra proteins spots at the basic part of the gels with molecular weights in the range of 25–15 kDa. The MALDI‐TOF‐MS of differential spots from acetone containing 10% TCA/0.07% 2‐mercaptoethanol precipitation method and absolute ethanol containing 10mM DTT indicated no similarity, ruling out the possibility that the two clusters shown represent identical proteins. The described method is easy in implementation, chemicals used are less toxic and proteins are easier to resuspend therefore presents an additional choice to implement towards finding the optimum method for extraction.     

Mass spectrometric characterization of the zein protein composition in maize flour extracts upon protein separation by SDS‐PAGE and 2D gel electrophoresis

Highly homogenous α zein protein was isolated from maize kernels in an environment‐friendly process using 95% ethanol as solvent. Due to the polyploidy and genetic polymorphism of the plant source, the application of high resolution separation methods in conjunction with precise analytical methods, such as MALDI‐TOF‐MS, is required to accurately estimate homogeneity of products that contain natural zein protein. The α zein protein product revealed two main bands in SDS‐PAGE analysis, one at 25 kDa and other at 20 kDa apparent molecular mass. Yet, high resolution 2DE revealed approximately five protein spot groups in each row, the first at ca. 25 kDa and the second at ca. 20 kDa. Peptide mass fingerprinting data of the proteins in the two dominant SDS‐PAGE bands matched to 30 amino acid sequence entries out of 102 non‐redundant data base entries. MALDI‐TOF‐MS peptide mapping of the proteins from all spots indicated the presence of only α zein proteins. The most prominent ion signals in the MALDI mass spectra of the protein mixture of the 25 kDa SDS gel band after in‐gel digestion were found at m/z 1272.6 and m/z 2009.1, and the most prominent ion signals of the protein mixture of the 20 kDa band after in‐gel digestion were recorded at m/z 1083.5 and m/z 1691.8. These ion signals have been found typical for α zein proteins and may serve as marker ion signals which upon chymotryptic digestion reliably indicate the presence of α zein protein in two hybrid corn products.     

Cationic detergents enable the separation of membrane proteins of Plasmodium falciparum-infected erythrocytes by 2D gel electrophoresis

The intraerythrocytic stage of Plasmodium falciparum alters the characteristics of its host cell by exporting selected plasmodial proteins. Although it is clear that the physicochemical and immunobiological properties of the host cell are modulated during parasite development, the involved plasmodial proteins and their mode of action are not completely known. Using cetyltrimethylammonium bromide (CTAB) or benzyldimethyl-n-hexadecylammonium chloride (16-BAC) for the first dimension and SDS for the second dimension, we separated proteins from membranes of human erythrocytes and of erythrocytes infected with the malaria parasite P. falciparum. Protein spots were analyzed by MALDI-TOF/TOF MS and annotated in respective 2D master gels. By using the alternative 2D approach, characteristic host cell membrane proteins and, more importantly, membrane-associated and exported plasmodial proteins were identified that might play a role in parasite-induced host cell modulation.     

Detection of recombinant growth hormone in human plasma by a 2‐D PAGE method

Human growth hormone (GH) has several central metabolic functions including bone growth in childhood, and its anabolic and lipolytic effects in particular are assumed reasons for the abuse of GH by athletes. Human endogenous GH consists of a main 22 kDa variant and several isoforms. In contrast, recombinant GH consists of only one variant being identical to the main endogenous isoform. The method presented here separates different isoforms by 2‐D PAGE after isolation of GH from plasma using an immunoaffinity purification system. While samples containing endogenous GH yield up to four isoforms, samples with recombinant GH contain the main 22 kDa spot only. Normalized spot volumes (NSV) are calculated after addition of an internal standard and a discrimination limit was determined at 0.52 for the NSV of the main 22 kDa spot. Above this value, samples containing endogenous GH show at least the main 22 kDa isoform and the 20 kDa splice variant. In contrast, samples with a NSV >0.52 and only one spot are suspicious to contain recombinant GH. This method detects discrete isoforms of GH from plasma and discriminates endogenous GH from its recombinant analog, which makes it useful for doping control purposes.     

Comparative proteomic analysis of different Toxoplasma gondii genotypes by two‐dimensional fluorescence difference gel electrophoresis combined with mass spectrometry

Toxoplasma gondii is a protozoan parasite infecting almost all warm‐blooded animals and humans. There are three infective stages of T. gondii: the tachyzoites, the bradyzoites, and the oocysts. The tachyzoite is a rapidly multiplying stage and the main pathogenic factor. In North America and Europe, T. gondii is consisted of four major clonal lineages (namely Types I, II, III, and Type 12). In this study, we explored the proteomic profiles of different genotypes (Type I‐RH strain, Type II‐PRU strain, Type II‐TgQHO strain, and ToxoDB 9‐TgC7 strain) of T. gondii tachyzoites by using 2D DIGE combined with MALDI‐TOF MS. Totally, 110 differentially abundant protein spots were selected. Of these, 98 spots corresponding to 56 proteins from T. gondii were successfully identified. These included surface antigen (SAG1), heat shock protein 70 (Hsp 70), disulfide isomerase, coronin, heat shock protein 60 (Hsp 60), pyruvate kinase, receptor for activated C kinase 1, and peroxiredoxin. Gene ontology enrichment analysis revealed that most of the differentially abundant proteins were involved in biological regulation, metabolic process, response to stress, binding, antioxidant activity, and transporter activity. According to the KEGG metabolic pathway maps of T. gondii, some identified proteins were involved in the glycolytic/gluconeogenesis pathway. The present study identified differentially abundant proteins among different genotypes of T. gondii and these findings have implications for the better understanding of the phenotypic differences among the examined T. gondii genotypes, which in turn may contribute to the better control of toxoplasmosis.     

Identification of candidate biomarkers in ovarian cancer serum by depletion of highly abundant proteins and differential in‐gel electrophoresis

Ovarian cancer is the fifth leading cause of cancer death for women in the US, yet survival rates are over 90% when it is diagnosed at an early stage, highlighting the need for biomarkers for early detection. To enhance the discovery of tumor‐specific proteins that could represent novel serum biomarkers for ovarian cancer, we depleted serum of highly abundant proteins which can mask the detection of proteins present in serum at low concentrations. Three commercial immunoaffinity columns were used in parallel to deplete the highly abundant proteins in serum from 60 patients with serous ovarian carcinoma and 60 non‐cancer controls. Medium and low abundance serum proteins from each serum pool were then evaluated by the quantitative proteomic technique of differential in‐gel electrophoresis. The number of protein spots that were elevated in ovarian cancer sera by at least twofold ranged from 36 to 248, depending upon the depletion and separation methods. From the 33 spots picked for MS analysis, nine different proteins were identified, including the novel candidate ovarian cancer biomarkers leucine‐rich α2 glycoprotein‐1 and ficolin 3. Western blotting validated the relative increases in serum protein levels for three of the proteins identified, demonstrating the utility of this approach for the identification of novel serum biomarkers for ovarian cancer.     

Efficient extraction of proteins from recalcitrant plant tissue for subsequent analysis by two‐dimensional gel electrophoresis

Protein extraction for two‐dimensional electrophoresis from tissues of recalcitrant species is quite problematic and challenging due to the low protein content and high abundance of contaminants. Proteomics in Shorea robusta is scarcely conducted due to the lack of a suitable protein preparation procedure. To establish an effective protein extraction protocol suitable for two‐dimensional electrophoresis in Shorea robusta, four procedures (borate buffer/trichloroacetic acid extraction, organic solvent/trichloroacetic acid precipitation, sucrose/Tris/phenol, and organic solvent/phenol/sodium dodecyl sulfate) were evaluated. Following these, proteins were isolated from mature leaves and were analyzed for proteomics, and also for potential contaminants, widely reported to hinder proteomics. The borate buffer/trichloroacetic acid extraction had the lowest protein yield and did not result in any banding even in one‐dimensional electrophoresis. In contrast, organic solvent/phenol/sodium dodecyl sulfate extraction allowed the highest protein yield. Moreover, during proteomics, organic solvent/phenol/sodium dodecyl sulfate extracted protein resolved the maximum number (144) of spots. Further, when proteins were evaluated for contaminants, significant (77–95%) reductions in the nucleic acids, phenol, and sugars were discernible with refinement in extraction procedure. Accumulated data suggested that the organic solvent/phenol/sodium dodecyl sulfate extraction was the most effective protocol for protein isolation for proteomics of Shorea robusta and can be used for plants that have a similar set of contaminants.     

Novel application of Ag nanoclusters in fluorescent imaging of human serum proteins after native polyacrylamide gel electrophoresis (PAGE)

We have developed a novel application for DNA oligonucleotide-stabilized Ag nanoclusters in fluorescent imaging of human serum proteins after native polyacrylamide gel electrophoresis (PAGE). Oligonucleotide-stabilized Ag nanoclusters were used as fluorescent probes for direct detection of proteins after native PAGE. Some relatively low-abundance proteins, such as α-1-antichymotrypsin (ACT) and α-2-glycoprotein 1, zinc (ZAG) were easily detected by oligonucleotide-stabilized Ag nanocluster-based fluorescent imaging and identified by MS and MS/MS techniques, without the need of expensive antibodies or tedious immunoassay procedures. The pH condition for the oligonucleotide-stabilized Ag nanocluster solution was optimized and the possible mechanism of interaction between proteins and DNA oligonucleotide-stabilized Ag nanoclusters was analyzed. As a novel fluorescent detection method it is simple, fast, nontoxic and sensitive, and it shows great analytical potential in proteome research and in biochemistry.     

Mass spectrometry‐based proteomics of oxidative stress: Identification of 4‐hydroxy‐2‐nonenal (HNE) adducts of amino acids using lysozyme and bovine serum albumin as model proteins

Modification of proteins by 4‐hydroxy‐2‐nonenal (HNE), a reactive by‐product of ω6 polyunsaturated fatty acid oxidation, on specific amino acid residues is considered a biomarker for oxidative stress, as occurs in many metabolic, hereditary, and age‐related diseases. HNE modification of amino acids can occur either via Michael addition or by formation of Schiff‐base adducts. These modifications typically occur on cysteine (Cys), histidine (His), and/or lysine (Lys) residues, resulting in an increase of 156 Da (Michael addition) or 138 Da (Schiff‐base adducts), respectively, in the mass of the residue. Here, we employed biochemical and mass spectrometry (MS) approaches to determine the MS “signatures” of HNE‐modified amino acids, using lysozyme and BSA as model proteins. Using direct infusion of unmodified and HNE‐modified lysozyme into an electrospray quadrupole time‐of‐flight mass spectrometer, we were able to detect up to seven HNE modifications per molecule of lysozyme. Using nanoLC‐MS/MS, we found that, in addition to N‐terminal amino acids, Cys, His, and Lys residues, HNE modification of arginine (Arg), threonine (Thr), tryptophan (Trp), and histidine (His) residues can also occur. These sensitive and specific methods can be applied to the study of oxidative stress to evaluate HNE modification of proteins in complex mixtures from cells and tissues under diseased versus normal conditions.     

A rapid and simple 8‐quinolinol‐based fluorescent stain of phosphoproteins in polyacrylamide gel after electrophoresis

In order to obtain an easy and rapid protocol to visualize phosphoproteins in SDS‐PAGE, a fluorescent detection method named 8‐Quinolinol (8‐Q) stain is described. 8‐Q can form ternary complexes in the gel matrix contributed by the affinity of aluminum ion (Al³⁺) to the phosphate groups on the proteins and the metal chelating property of 8‐Quinolinol, exhibiting strong fluorescence in ultraviolet light. It can visualize as little as 4～8 ng of α‐casein and β‐casein, 16～32 ng of ovalbumin and κ‐casein which is more sensitive than Stains‐All but less sensitive than Pro‐Q Diamond. The protocol of 8‐Q requires only 70 min in 0.75 mm mini‐size or 1.0 mm large‐size gels with five changes of solutions without destaining step; Pro‐Q takes at least 250 min with 11 changes of solutions. In addition, the new method was confirmed by the study of dephosphorylation and LC‐MS/MS, respectively. The approach to visualize phosphoprotein utilizing 8‐Q could be an alternative to simplify the analytical operations for phosphoproteomics research.     

Capillary electrophoresis separation of human milk neutral and acidic oligosaccharides derivatized with 2‐aminoacridone

Human milk is a unique fluid in glycobiology due to the presence of many free structurally complex oligosaccharides emerging as important dietary factors during early life and having many biological and protective functions. Methods that allow accurate profiling of oligosaccharide mixtures in this complex biological fluid with quantification of the four known genetically determined groups are welcomed. A high‐voltage CE separation and detection at 254 nm of 17 neutral and acidic human milk oligosaccharide (HMO) standard along with lactose derivatized with 2‐aminoacridone, using a BGE containing 20% methanol as an organic modifier and borate, able to form on‐capillary anionic borate‐polyol complexes, is reported. This CE approach was able to separate both neutral HMOs and acidic HMOs, with the sialic acid residue, also in the presence of lactose in high content. This method was applied to the four secretory groups individually extracted by a rapid and simple preparative step. LODs were found ranging from ～50 to 700 fmol. We were able to measure HMO content also in the presence of excess fluorophore, or interference from proteins, peptides, salts, and other impurities normally present in this complex biological fluid. Overall, CE equipped with a UV detector is a common analytical approach and this simple CE separation offers high resolution and sensitivity for the differentiation of human milk samples related to genetic groups and days of lactation by considering that important changes in HMO content are a reflection of the lactation day.     

Application of two‐dimensional near‐infrared (2D‐NIR) correlation spectroscopy to the discrimination of three species of Dendrobium

The objective of this paper was to apply two‐dimensional (2D) near‐infrared (NIR) correlation spectroscopy to the discrimination of three species of Dendrobium. Generalized 2D‐NIR correlation spectroscopy was able to enhance spectral resolution, simplify the spectrum with overlapped bands and provide information about temperature‐induced spectral intensity variations that was hard to obtain from one‐dimensional NIR spectroscopy. The FT‐NIR spectra were measured over a temperature range of 30–140°C. The 2D synchronous and asynchronous spectra showed remarkable differences within the range of 5600–4750 cm⁻¹ between different species of Dendrobium. Copyright © 2009 John Wiley & Sons, Ltd.     

Characterization of crude oil biomarkers using comprehensive two‐dimensional gas chromatography coupled to tandem mass spectrometry

Oil samples from Recôncavo basin (NE Brazil), previously analyzed by traditional techniques such as gas chromatography coupled to tandem mass spectrometry, were evaluated using comprehensive two‐dimensional gas chromatography coupled to quadrupole mass spectrometry and comprehensive two‐dimensional gas chromatography coupled to tandem mass spectrometry along with simplified methods of samples preparation to evaluate the differences and advantages of these analytical techniques to better understand the development of the organic matter in this basin without altering the normal distribution of the compounds in the samples. As a result, the geochemical parameters calculated by comprehensive two‐dimensional gas chromatography coupled to tandem mass spectrometry described better the origin, maturity, and biodegradation of both samples probably by increased selectivity, resolution, and sensitivity inherent of the multidimensional technique. Additionally, the detection of the compounds such as, the C(14α‐) homo‐26‐nor‐17α‐hopane series, diamoretanes, nor‐spergulanes, C₁₉–C₂₆ A‐nor‐steranes and 4α‐methylsteranes resolved and detected by comprehensive two‐dimensional gas chromatography coupled to tandem mass spectrometry were key to classify and differentiate these lacustrine samples according to their maturity and deposition conditions.     

Searching for serum tumor markers for colorectal cancer using a 2‐D DIGE approach

Identification of specific protein markers for colorectal cancer (CRC) could provide a basis for its early diagnosis and detection, as well as clues to the molecular mechanisms governing cancer progression. In the present study, 2‐D DIGE coupled with MS was used to screen for biomarker candidates in the serum proteome of ten human CRC samples and ten healthy control samples. After pooling identical amounts of serum proteins (based on total protein concentration), albumin/IgG was depleted under partially denaturing conditions. Subsequently, the serum samples were labeled with three different CyDyes, and separated by 2‐D DIGE. After analysis with the biological variation analysis module of the DeCyder software, only three spots were found to be significantly elevated in all patient groups (with ratios from 1.52 to 9.08), whereas five spots were significantly down‐regulated in patients (with ratios from ?1.23 to ?10.21) (t‐test; p<0.05). Finally, two potential biomarkers, Transaldolase 1 and thyroid receptor interactor, were chosen for validation and analysis by ELISA with the serum of 30 CRC patients and 30 healthy controls. The serum levels of the two proteins correlated well with the 2‐D DIGE results. Thus, 2‐D DIGE approaches show great promise for biomarker discovery in CRC.     

Sequential double fluorescent detections of total proteins and phosphoproteins in SDS‐PAGE

A fluorescent staining technique, using selective chelation with fluorophore and metal ion to the phosphate groups of phosphoproteins in SDS‐PAGE is described. As a fluorescent dye and a metal ion, Fura 2 pentapotassium salt and Al³⁺ were employed, respectively. The staining method, Fura 2 stain, has sensitivities of 16–32 ng of α‐casein and β‐casein, 62 ng of ovalbumin, phosvitin, and κ‐casein using an ultraviolet transilluminator. Furthermore, Fura 2 stain is able to carry out continuative double detection of total proteins and phosphoproteins on the same gel within 3.5 h. Consequently, selective phosphoprotein and total protein detections could be obtained without other poststaining. Considering the low cost, simplicity, and speed, Fura 2 staining may provide great practicalities in routine phosphoproteomics research.