Electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) versus strong cation exchange (SCX) for fractionation of iTRAQ-labeled peptides

The iTRAQ technique is popular for the comparative analysis of proteins in different complex samples. To increase the dynamic range and sensitivity of peptide identification in shotgun proteomics, SCX chromatography is generally used for the fractionation of iTRAQ-labeled peptides before LC-MS/MS analysis. However, SCX suffers from clustering of similarly charged peptides and the need to desalt fractions. In this report, SCX is compared with the alternative ERLIC method for fractionating iTRAQ-labeled peptides. The simultaneous effect of electrostatic repulsion and hydrophilic interaction in ERLIC results in peptide elution in order of decreasing pI and GRAVY values (increasing polarity). Volatile solvents can be used. We applied ERLIC to iTRAQ-labeled peptides from rat liver tissue, and 2745 proteins and 30,016 unique peptides were identified with high confidence from three technical replicates. This was 12.9 and 49.4% higher, respectively, than was obtained using SCX. In addition, ERLIC is appreciably better at the identification of highly hydrophobic peptides. The results indicate that ERLIC is a more convenient and more effective alternative to SCX for the fractionation of iTRAQ-labeled peptides. Quantification data show that both SCX and ERLIC fractionation have no significant effect on protein quantification by iTRAQ.     

Development and application of a phosphoproteomic method using electrostatic repulsion-hydrophilic interaction chromatography (ERLIC), IMAC, and LC-MS/MS analysis to study Marek's Disease Virus infection

Marek's Disease (MD) is an avian neoplastic disease caused by Marek's Disease Virus (MDV). The mechanism of virus transition between the lytic and latent cycle is still being investigated; however, post-translational modifications, especially phosphorylation, have been thought to play an important role. Previously, our group has used strong cation exchange chromatography in conjunction with reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) to study the changes in global proteomic expression upon MDV infection (Ramaroson , M. F.; Ruby, J.; Goshe, M. B.; Liu , H.-C. S. J. Proteome Res. 2008, 7, 4346-4358). Here, we extend our study by developing an effective separation and enrichment approach to investigate the changes occurring in the phosphoproteome using electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) to fractionate peptides from chicken embryo fibroblast (CEF) digests and incorporating a subsequent IMAC enrichment step to selectively target phosphorylated peptides for LC-MS/MS analysis. To monitor the multidimensional separation between mock- and MDV-infected CEF samples, a casein phosphopeptide mixture was used as an internal standard. With LC-MS/MS analysis alone, no CEF phosphopeptides were detected, while with ERLIC fractionation only 1.2% of all identified peptides were phosphorylated. However, the incorporation of IMAC enrichment with ERLIC fractionation provided a 50-fold increase in the percentage of identified phosphopeptides. Overall, a total of 581 unique phosphopeptides were identified (p < 0.05) with those of the MDV-infected CEF sample containing nearly twice as many as the mock-infected control of which 11% were unique to MDV proteins. The changes in the phosphoproteome are discussed including the role that microtubule-associated proteins may play in MDV infection mechanisms.     

Hydrophilic interaction chromatography reduces the complexity of the phosphoproteome and improves global phosphopeptide isolation and detection

The diversity and complexity of proteins and peptides in biological systems requires powerful liquid chromatography-based separations to optimize resolution and detection of components. Proteomics strategies often combine two orthogonal separation modes to meet this challenge. In nearly all cases, the second dimension is a reverse phase separation interfaced directly to a mass spectrometer. Here we report on the use of hydrophilic interaction chromatography (HILIC) as part of a multidimensional chromatography strategy for proteomics. Tryptic peptides are separated on TSKgel Amide-80 columns using a shallow inverse organic gradient. Under these conditions, peptide retention is based on overall hydrophilicity, and a separation truly orthogonal to reverse phase is produced. Analysis of tryptic digests from HeLa cells yielded numbers of protein identifications comparable to that obtained using strong cation exchange. We also demonstrate that HILIC represents a significant advance in phosphoproteomics analysis. We exploited the strong hydrophilicity of the phosphate group to selectively enrich and fractionate phosphopeptides based on their increased retention under HILIC conditions. Subsequent IMAC enrichment of phosphopeptides from HILIC fractions showed better than 99% selectivity. This was achieved without the use of derivatization or chemical modifiers. In a 300-microg equivalent of HeLa cell lysate we identified over 1000 unique phosphorylation sites. More than 700 novel sites were added to the HeLa phosphoproteome.     

固化金属离子亲和层析富集磷酸化多肽方法的选择及优化

在磷酸化蛋白质组学研究中,根据是否需要对待富集样品进行甲酯化处理,可将固化金属离子亲和层析（IMAC）方法分为两类,即需要甲酯化处理的IMAC方法（ME—IMAC）和不需要甲酯化处理的IMAC方法（Non—ME—IMAC）。要实现对磷酸化多肽的有效富集和鉴定,就必须对富集方法进行选择和优化。利用基质辅助激光解析离子化串联飞行时间质谱（MALDI—TOF—MS）对两种方法富集的磷酸化多肽进行了比较研究。结果表明,ME—IMAC方法容易发生样品丢失,质谱结果的分析也比较复杂,而Non—ME—IMAC方法则不仅操作简单而且富集效果理想。另外,优化了Non—ME—IMAC方法的实验条件,指出最佳的结合溶液是8％ACN／0．3％TFA,最佳的洗脱溶液是0．1mol／LEDTA（pH值8．0）,从而建立了一套完整而简单有效的磷酸化多肽富集方法。     

Current methods for phosphoprotein isolation and enrichment

The phosphorylation of proteins is a central paradigm of signal transduction. The substitution of neutral hydroxyl groups of serine, threonine and tyrosine with a negatively charged phosphate group alters the physicochemical and immunogenic properties of the protein, which then can be used to isolate these isoforms. In the last decades several different techniques were applied, attempting to selectively enrich protein populations with this post-translational modification. This review aims to give an overview on the arsenal of available methods to extract phosphoproteins focusing on chromatographic approaches.     

Specific phosphopeptide enrichment with immobilized titanium ion affinity chromatography adsorbent for phosphoproteome analysis

The elucidation of protein post-translational modifications, such as phosphorylation, remains a challenging analytical task for proteomic studies. Since many of the proteins targeted for phosphorylation are low in abundance and phosphorylation is typically substoichiometric, a prerequisite for their identification is the specific enrichment of phosphopeptide prior to mass spectrometric analysis. Here, we presented a new method termed as immobilized titanium ion affinity chromatography (Ti (4+)-IMAC) for enriching phosphopeptides. A phosphate polymer, which was prepared by direct polymerization of monomers containing phosphate groups, was applied to immobilize Ti (4+) through the chelating interaction between phosphate groups on the polymer and Ti (4+). The resulting Ti (4+)-IMAC resin specifically isolates phosphopeptides from a digest mixture of standard phosphoproteins and nonphosphoprotein (BSA) in a ratio as low as 1:500. Ti (4+)-IMAC was further applied for phosphoproteome analysis of mouse liver. We also compared Ti (4+)-IMAC to other enrichment methods including Fe (3+)-IMAC, Zr (4+)-IMAC, TiO 2 and ZrO 2, and demonstrate superior selectivity and efficiency of Ti (4+)-IMAC for the isolation and enrichment of phosphopeptides. The high specificity and efficiency of phosphopeptide enrichment by Ti (4+)-IMAC mainly resulted from the flexibility of immobilized titanium ion with spacer arm linked to polymer beads as well as the specific interaction between immobilized titanium ion and phosphate group on phosphopeptides.     

Enhanced phosphopeptide isolation by Fe(III)-IMAC using 1,1,1,3,3,3-hexafluoroisopropanol

IMAC can be used to selectively enrich phosphopeptides from complex peptide mixtures, but co-retention of acidic peptides together with the failure to retain some phosphopeptides restricts the general utility of the method. In this study Fe(III)-IMAC was qualitatively and quantitatively assessed using a panel of phosphopeptides, both synthetic and derived from proteolysis of known phosphoproteins, to identify the causes of success and failure in the application of this technique. Here we demonstrate that, as expected, peptides with a more acidic amino acid content are generally more efficiently purified and detected by MALDI-MS after Fe(III)-IMAC than those with a more basic content. Modulating the loading buffer used for Fe(III)-IMAC significantly affects phosphopeptide binding and suggests that conformational factors that lead to steric hindrance and reduced accessibility to the phosphate are important. The use of 1,1,1,3,3,3-hexafluoroisopropanol is shown here to significantly improve Fe(III)-IMAC enrichment and subsequent detection of phosphopeptides by MALDI-MS.     

Identification of novel phosphorylation sites on Xenopus laevis Aurora A and analysis of phosphopeptide enrichment by immobilized metal-affinity chromatography

Mass spectrometric analysis of proteolytically derived phosphopeptides has developed into a widespread technique for the identification of phosphorylated amino acids. Using liquid chromatography-electrospray ionization tandem mass spectrometry, 14 phosphorylation sites were identified on Xenopus laevis His6-Aurora A, a highly conserved regulator of centrosome maturation and cell division. These included seven novel phosphorylation sites, Ser-12, Thr-21, Thr-103, Ser-116, Thr-122, Tyr-155, and Thr-294, as well as the previously identified regulatory sites, Ser-53, Thr-295, and Ser-349. The identification of these novel phosphorylation sites will be important for future studies aimed at elucidating the mechanisms of Aurora A regulation by phosphorylation. Furthermore, we demonstrate that a "kinase-inactive" mutant of Aurora A, K169R, still retains 10% of activity of the wild-type enzyme in vitro along with occupancy of Thr-295 and Ser-12. However, mutation of Asp-281 to Ala completely abolishes activity of the enzyme and should therefore be used preferentially as a genuine kinase-dead construct. Because of the abundance of phosphorylated residues on His6-Aurora A, we found this protein to be an ideal tool for the characterization of immobilized metal-affinity chromatography (IMAC) as a method for phosphopeptide enrichment from complex mixtures. We present a detailed analysis of the binding and elution properties of both the phosphopeptides and unphosphorylated peptides of His6-Aurora A to Fe3+-IMAC before and after methyl esterification. Moreover, we demonstrate a significant difference in enrichment of phosphopeptides when different resins are used for Fe3+-IMAC and characterize the strengths and limitations of this methodology for the study of phosphoproteomics.     

Optimization of phosphopeptide elution conditions in immobilized Fe(III) affinity chromatography

While immobilized metal affinity chromatography (IMAC) has been widely used for affinity purification of phosphopeptides, the technique suffers from insufficient specificity. Therefore, there is an urgent need for IMAC optimization to yield the selectivity and sensitivity that is required for more challenging analyses. Recently, 2,5-dihydroxybenzoic acid (DHB) and phosphoric acid mixture has been reported as an efficient IMAC eluant. The disadvantage of DHB is that is not suitable for electrospray ionization-mass spectrometry. While further developing the IMAC elution protocol to overcome this problem, we noticed that DHB is not necessary and found a novel combination of phosphoric acid and acetonitrile to be more efficient. The purification efficacy of the novel protocol is superior to all previously described methods, while still being compatible with the most commonly used mass-spectrometric techniques in phosphoproteomics.     

A comparative study of clustering methods for molecular data

The research aim is to use three clustering technologies for establishing molecular data model of large size sets by comparison between low energy samples (LES) and local molecular samples (LMS). Hierarchical cluster of multi-level tree distance relation, competitive learning network of similar inputs falling into the same cluster and topological SOM are used to analyze 6,242 LES and 5,000 LMS. Our experiments show that in SOM, there are 24 to 25 Davies-Boulding clustering index and color map cluster units in the LES more than 10 to 12 in the LMS, which is consistent with the results of hierarchical cluster and competitive learning network in the rough. The hierarchical cluster reflects the biggest inter-cluster distance about 30 for the LES is far larger than that of LMS about 10. The intra-cluster distance of LES about 15 is also far bigger than that of LMS about 3. In SOM, there are more cluster borders of high values (black) reflecting large distance and more clusters in the D-matrix and U-matrix of LES than that of LMS, due to the biggest standard deviation range from -8 to 10 of samples feature of the LES is bigger than that of LMS from -2.5 to 2.5.     

Evaluation of four phosphopeptide enrichment strategies for mass spectrometry-based proteomic analysis

Information about phosphorylation status can be used to prioritize and characterize biological processes in the cell. Various analytical strategies have been proposed to address the complexity of phosphorylation status and comprehensively identify phosphopeptides. In this study, we evaluated four strategies for phosphopeptide enrichment, using titanium dioxide (TiO₂) and Phos-tag ligand particles from in-gel or in-solution digests prior to mass spectrometry-based analysis. Using TiO₂ and Phos-tag magnetic beads, it was possible to enrich phosphopeptides from in-gel digests of phosphorylated ovalbumin separated by Phos-tag SDS-PAGE or in-solution serum digests, while minimizing non-specific adsorption. The tip-column strategy with TiO₂ particles enabled enrichment of phosphopeptides from in-solution digests of whole-cell lysates with high efficiency and selectivity. However, the tip-column strategy with Phos-tag agarose beads yielded the greatest number of identified phosphopeptides. The strategies using both types of tip columns had a high degree of overlap, although there were differences in selectivity between the identified phosphopeptides. Together, our results indicate that multi-enrichment strategies using TiO₂ particles and Phos-tag agarose beads are useful for comprehensive phosphoproteomic analysis.     

A procedure for the enrichment and isolation of Halobacterium

A procedure for the specific enrichment and isolation of species of the genus Halobacterium was designed, based on the ability of Halobacterium cells to grow anaerobically by fermentation of l-arginine. None of the other genera of neutrophilic halophilic Archaea tested grew fermentatively on arginine. Using anaerobic enrichments in the presence of arginine, representatives of the genus Halobacterium were consistently isolated from saltern crystallizer ponds in Eilat (Israel) and San Francisco Bay (California), environments in which Halobacterium represents only a very small fraction of the halophilic archaeal community.     

A comparative study in methods of cryoprotection for human semen

A comparative study using either glycerol or an egg yolk-citrate-glycerol mix for cryoprotection under the conditions described showed the latter to give a significantly better post-thaw motility. The greatest drop was noted within the first hour, suggesting that freezability of a sample could be judged accurately by rethawing at that time.Controlled studies using scanning electron microscopy clearly showed a greater head disruption after freezing with glycerol and looped tails after centrifugation, which could account for some of the findings of the first part of the study.These findings are reviewed and it is recommended that seminal samples are not centrifuged and that a complex medium be employed for seminal freezing and storing.     

Comparative study of enrichment methods for the isolation of autotrophic nitrifying bacteria from soil, estuarine and marine sediments

Abstract A comparative study has been undertaken to determine the efficiency of methods for the enrichment and isolation of autotrophic nitrifying bacteria from soils and estuarine and marine sediments. Chemostat enrichments proved to be the most efficient means of isolating autotrophic NH⁺₄ oxidisers whereas NO⁻₂ oxidising bacteria were never successfully enriched by this method. In contrast, gel enrichment and traditional batch culture enrichments of nitrifying bacteria were comparatively time consuming procedures and the degree of enrichment obtained for NH⁺₄ oxidising bacteria never approached that obtained with continuous culture enrichments. Gel enrichments, however, because they have continuous physicochemical gradients provide qualitative advantages in that morphologically distinct types of nitrifying bacteria can be isolated from the same gel.     

Hair removal methods: A comparative study for dermoscopy images

Removal and restoration of hair and hair-like regions within skin lesion images is needed so features within lesions can be more effectively analyzed for benign lesions, cancerous lesions, and for cancer discrimination. This paper refers to “melanoma texture” as a rationale for supporting the need for the proposed hair detection and repair techniques, which incompletely represents why hair removal is an important operation for skin lesion analysis. A comparative study of the state-of-the-art hair-repaired methods with a novel algorithm is also proposed by morphological and fast marching schemes. The hair-repaired techniques are evaluated in terms of computational, performance and tumor-disturb patterns (TDP) aspects. The comparisons have been done among (i) linear interpolation, inpainting by (ii) non-linear partial differential equation (PDE) and (iii) exemplar-based repairing techniques. The performance analysis of hair detection quality, was based on the evaluation of the hair detection error (HDE), quantified by statistical metrics and manually used to determine the hair lines from a dermatologist as the ground truth. The results are presented on a set of 100 dermoscopic images. For the two characteristics measured in the experiments the best method is the fast marching hair removal algorithm (HDE: 2.98%, TDP: 4.21%). This proposed algorithm repaired the texture of the melanoma, which becomes consistent with human vision. The comparisons results obtained, indicate that hair-repairing algorithm based on the fast marching method achieve an accurate result.     

A comparative study of the interaction of two structurally analogue ruthenium(II) complexes with DNA

The binding of the ruthenium(II) complexes of [Ru(bpy)2(CAIP)]Cl2 and [Ru(bpy)2(HCIP)]Cl2 (where bpy=2,2'-bipyridine, CAIP=4-carboxyl-imidado[4,5-f][1,10]-phenanthroline, HCIP=3-hydroxyl-4-carboxyl-imidado[4,5-f][1,10]-phenanthroline) to calf thymus DNA (ct-DNA) has been investigated with UV-visible and emission spectroscopy, steady-state emission quenching, and viscosity measurements. The experimental results indicate that the two complexes bind to ct-DNA through an intercalative mode and [Ru(bpy)2(HCIP)]2+ intercalates into DNA more deeply than [Ru(bpy)2(CAIP)]2+ does.     

Coupling flash liquid chromatography with mass spectrometry for enrichment and isolation of milk oligosaccharides for functional studies

Mass spectrometry has been coupled with flash liquid chromatography to yield new capabilities for isolating nonchromophoric material from complicated biological mixtures. A flash liquid chromatography/tandem mass spectrometry (LC/MS/MS) method enabled fraction collection of milk oligosaccharides from biological mixtures based on composition and structure. The method is compatible with traditional gas pressure-driven flow flash chromatography widely employed in organic chemistry laboratories. The online mass detector enabled real-time optimization of chromatographic parameters to favor separation of oligosaccharides that would otherwise be indistinguishable from coeluting components with a nonspecific detector. Unlike previously described preparative LC/MS techniques, we have employed a dynamic flow connection that permits any flow rate from the flash system to be delivered from 1 to 200 ml/min without affecting the ionization conditions of the mass spectrometer. A new way of packing large amounts of graphitized carbon allowed the enrichment and separation of milligram quantities of structurally heterogeneous mixtures of human milk oligosaccharides (HMOs) and bovine milk oligosaccharides (BMOs). Abundant saccharide components in milk, such as lactose and lacto-N-tetraose, were separated from the rarer and less abundant oligosaccharides that have greater structural diversity and biological functionality. Neutral and acidic HMOs and BMOs were largely separated and enriched with a dual binary solvent system.     

A comparative study of proteolysis methods for the measurement of 3-nitrotyrosine residues: enzymatic digestion versus hydrochloric acid-mediated hydrolysis

A common approach for the quantification of 3-nitrotyrosine (NY) in routine analyses relies on the cleavage of peptide bonds in order to release the free amino acids from proteins in tissues or fluids. NY is usually monitored by either GC-MS(/MS) or LC-MS/MS techniques. Various proteolysis methods have been employed to combine digestion efficiency with prevention of artifactual nitration of tyrosine. However, so far, no study was designed to compare the HCl-based hydrolysis method with enzymatic digestion in terms of reliability for the measurement of NY. The present work addresses the digestion efficiency of BSA using either 6M HCl, pronase E or a cocktail of enzymes (pepsin, pronase E, aminopeptidase, prolidase) developed in our laboratory. The HCl-based hydrolysis leads to a digestion yield of 95%, while 25 and 75% are achieved with pronase E and the cocktail of enzymes, respectively. These methods were compared in terms of NY measurement and the results indicate that a prior reduction of the disulfide bonds ensures a reliable quantification of NY. We additionally show that the enzyme efficacy is not altered when the digestion is carried out in the presence of BSA with a high content of NY.     

A comparative study of crested gibbons (Nomascus)

Crested gibbons (Nomascus) are in the rarest genus of the family Hylobatidae, with the Hainan gibbon (Nomascus hainanus) being the rarest primate in the world. In the past, the number of species in this genus has been at the center of much controversy, in part, because their color changes during immaturity as well as other factors, such as physical similarities in genitalia, creating difficulties in accurately determining the sex of individuals. Furthermore, owing to their rarity, illusiveness, and the rough terrain that comprises their native habitat, Nomascus is one of the least studied Hylobatidae. This article represents the most comprehensive dissemination of visual characteristics of the genus Nomascus to assist in the accurate identification of captive and wild crested gibbons. Through differences in pelage color, skeletal anatomy, dentition, vocalizations, behavior, distribution, and genetic studies, we are able to determine more accurately whether or not a subspecies should be elevated to species level. From the current data, there are six species and one subspecies in the genus Nomascus. However, reports of a recently identified light-cheeked gibbon (Nomascus sp.) in northeast Cambodia, Central Vietnam, and South Lao PDR, will add additional taxa to this genus.