Future potential of targeted component analysis by multidimensional liquid chromatography-mass spectrometry

Multidimensional liquid chromatography (MDLC) may be used in either (i) the profiling mode where it is the objective to fractionate all components in a mixture or (ii) the targeted component mode in which it is the objective to determine specific analytes. This paper focuses on targeted component analysis from complex mixtures, addressing the critical operations of analyte selection and transport from the first to the second dimension. Although the physical operation of switching a component into the second dimension with computer controlled valving is simple, it is shown that changes in analyte retention time and peak width with column age and fouling are a serious problem. The analyte moves out of the preselected time window for valve switching and quantitation is compromised in the second dimension. It is proposed that a solution to the “drifting peak” phenomenon in targeted component analysis is to use binary mobility elution in the first dimension. Binary mobility refers to those systems, such as affinity chromatography, in which analyte mobility is generally either 0 or 1 relative to mobile phase velocity. Coupling these binary changes in analyte mobility in the first dimension with valve switching eliminates the “drifting peak” phenomenon. In addition, it is shown that a wide time window may be used in affinity separations without compromising the separation or accumulating contaminants. Several cases are described in which immunosorbents were used with reversed phase columns to provide quantitative targeted component analyses from complex mixtures.     

多维液相色谱-质谱用于酶解猪血蛋白中活性肽的研究

以强阳离子交换柱（SCX）为一维色谱柱,反相柱（RP）为二维色谱柱,采用在线捕集接口形式,通过10通阀连接一、二维色谱柱,构建了二维液相色谱分离系统。将该系统用于酶解猪血蛋白中对血管紧缩素Ⅰ转移酶（ACE）具有活性抑制作用的肽进行分离、鉴定,共检测出104个组分。收集一维馏分,离线注入LC—MS,鉴定出其中含有SAL、DKF、ESF、STVL及FESF5个小肽。     

Limitations of current proteomics technologies

Application of proteomics technologies in the investigation of biological systems creates new possibilities in the elucidation of biopathomechanisms and the discovery of novel drug targets and early disease markers. A proteomic analysis involves protein separation and protein identification as well as characterization of the post-translational modifications. Proteomics has been applied in the investigation of various disorders, like neurological diseases, and the application has resulted in the detection of a large number of differences in the levels and the modifications of proteins between healthy and diseased states. However, the current proteomics technologies are still under development and show certain limitations. In this article, we discuss the major drawbacks and pitfalls of proteomics we have observed in our laboratory and in particular during the application of proteomics technologies in the investigation of the brain.     

Proteomics of snake venoms from Elapidae and Viperidae families by multidimensional chromatographic methods

Snake venoms contain a large number of biologically active substances and the venom components are very useful for pharmaceutical applications. Our goal is to separate and identify components of snake venoms in ten snake species from the Elapidae and Viperidae families using multidimensional chromatographic methods. The multidimensional chromatographic methods include reversed-phase high-performance liquid chromatography (RP-HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), lab-on-a-chip, two-dimensional electrophoresis (2-DE), and mass spectrometry. The venoms of eight snake species demonstrated major differences in hydrophobicity, molecular weight separations, and 2-DE protein distribution patterns. The 2-DE images showed major differences between families, within each family and even between the same species. Venoms of the Elapidae family showed many basic proteins with a wide range of molecular weights, while venoms of the Viperidae family showed wide ranges of pI and molecular weights, especially for Trimeresurus sp. The multidimensional chromatographic methods revealed specific differences in venom proteins intra-species as well as between species and families. We have isolated and identified proteins that may be unique for each species for further studies in the proteome of snake venoms and their potentially use in the pharmaceutical applications.     

Comprehensive two-dimensional liquid chromatography coupled with mass spectrometry

In this review, instrumental aspects of comprehensive two-dimensional liquid chromatography coupled with mass spectrometry are presented. The milestones of LC×LC are briefly summarized. Instrument configuration, selection of experimental conditions, the different interfaces used in the system and the current applications of LC×LC–MS systems are described.     

High-efficiency liquid chromatography–mass spectrometry separations with 50 mm, 250 mm,and 1 m long polymer-based monolithic capillary columns for the characterization of complex proteolytic digests

In this study, high-efficiency LC–MS/MS separations of complex proteolytic digests are demonstrated using 50 mm, 250 mm, and 1 m long poly(styrene-co-divinylbenzene) monolithic capillary columns. The chromatographic performance of the 50 and 250 mm monoliths was compared at the same gradient steepness for gradient durations between 5 and 150 min. The maximum peak capacity of 400 obtained with a 50 mm column, increased to 485 when using the 250 mm long column and scaling the gradient duration according column length. With a 5-fold increase in column length only a 20% increase in peak capacity was observed, which could be explained by the larger macropore size of the 250 mm long monolith. When taking into account the total analysis time, including the dwell time, gradient time and column equilibration time, the 50 mm long monolith yielded better peptide separations than the 250 mm long monolithic column for gradient times below 80 min (n_c = 370). For more demanding separation the 250 mm long monolith provided the highest peak production rate and consequently higher sequence coverage. For the analysis of a proteolytic digest of Escherichia coli proteins a monolithic capillary column of 1 m in length was used, yielding a peak capacity of 1038 when applying a 600 min gradient.     

Two dimensional separations of human urinary protein digest using a droplet-interfaced platform

For highly complex mixtures, coelution is a common phenomenon in chromatography. A great deal of resolution is hidden in coelution, and lost due to inevitable molecular diffusion during sample transfer. The molecular diffusion may lead to band broadening and remix of separated peaks, which cause degradation of achievable resolution. In this study, we introduced droplet microfluidics as a high performance sample transfer tool in two dimensional nanoflow liquid chromatography–capillary electrophoresis separation of a human urine sample. The fine fractionation capability and sampling completeness enabled by the droplet-interface demonstrated the 2D system’s usefulness in high-resolution mapping of real world biological samples.     

The dimensionality of chromatographic separations

The box-counting or capacity dimension algorithm, known from the fractal mathematics literature, is used to measure the dimensionality D of chromatographic separation techniques for any number of dimensions. It is shown that D has limit properties that match Giddings' sample dimensionality s. D values are shown to be sensitive to the uniformity of peak spacing. A number of examples are given where D is calculated for various limits in one- and two-dimensional separations and for heart-cutting separations. The use of D as a quantitative measure of multidimensional orthogonality is suggested as D, due to the scale-free nature, is not dependent on the effective separation area. The connection to statistical peak overlap theory is discussed.     

Diagonal chromatographic selection of cysteinyl peptides modified with benzoquinones

The derivatization of cysteine-containing peptides with benzoquinone compounds is rapid, quantitative and specific in acidic media. The conversion of cysteines into hydrophobic benzoquinone-adducted residues in peptides is used here to alter the chromatographic properties of cysteinyl peptides during liquid chromatography separation. The benzoquinone derivatization is shown to allow the accurate selection of cysteine-containing peptides of bovine serum albumin tryptic digest by diagonal reversed-phase chromatography, which consists of one primary and a series of secondary identical liquid chromatographic separations, before and after a cysteinyl-targeted modification of the peptides by benzoquinone compounds. Figure Diagonal chromatographic selection of cysteinyl peptides modified with benzoquinones Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Comprehensive two-dimensional liquid chromatographic analysis of rooibos (Aspalathus linearis) phenolics

Rooibos tea is an unique beverage prepared from unfermented and fermented plant material of the endemic Cape fynbos plant, Aspalathus linearis. The well-known health-promoting benefits of rooibos are partly attributed to its phenolic composition. Detailed investigation of the minor phenolic constituents of rooibos is, however, hampered by the limitations associated with conventional HPLC methods used for its analysis. In this study, the applicability of comprehensive two-dimensional liquid chromatographic methods for the in-depth analysis of rooibos phenolics was investigated. Phenolic compounds were separated according to polarity by hydrophilic interaction chromatography (HILIC) in the first dimension, whilst reversed-phase liquid chromatography (RP-LC) provided separation according to hydrophobicity in the second dimension. Ultraviolet photodiode array and electrospray ionisation mass spectrometry were used to identify phenolic compounds. Comprehensive HILIC × RP-LC demonstrated its applicability for the analysis of a diverse range of phenolic compounds in unfermented and fermented rooibos samples, in which large qualitative differences in the phenolic composition were established. The combination of these orthogonal separations provided a significant improvement in resolution, as exemplified by practical peak capacities in excess of 2000 and 500 for off-line and on-line methods, respectively.     

Estimating optimal time for fast chromatographic separations

The term t_{min cc} provides a ready estimate of the shortest time that can be obtained by “column cutting” for baseline resolution of two components showing excess chromatographic resolution. While actual column cutting is impractical, the t_{min cc} value is shown to be closely related to the minimum separation time obtainable by adjusting other parameters such as flow rate, mobile phase composition, and temperature, affording scientists interested in the development of fast chromatographic separations a convenient tool for estimating the minimum separation time that can be obtained by modifying a given method development screening result. Furthermore, the relationship between t_{min cc} and the minimum separation time obtainable by adjusting other parameters is shown to be dependent on the speed of the screening method, with aggressive screening gradients affording t_{min cc} estimates that match the actual minimum separation time, and “lazy” screening gradients affording t_{min cc} values that overestimate minimum separation time. Consequently, the analysis of the relationship between t_{min cc} and actual minimum separation time may be a useful tool for determining the “fitness” of method development screening methods.     

Hydrophilic interaction liquid chromatography (HILIC) in proteomics

In proteomics, nanoflow multidimensional chromatography is now the gold standard for the separation of complex mixtures of peptides as generated by in-solution digestion of whole-cell lysates. Ideally, the different stationary phases used in multidimensional chromatography should provide orthogonal separation characteristics. For this reason, the combination of strong cation exchange chromatography (SCX) and reversed-phase (RP) chromatography is the most widely used combination for the separation of peptides. Here, we review the potential of hydrophilic interaction liquid chromatography (HILIC) as a separation tool in the multidimensional separation of peptides in proteomics applications. Recent work has revealed that HILIC may provide an excellent alternative to SCX, possessing several advantages in the area of separation power and targeted analysis of protein post-translational modifications. Figure Artistic impression of the HILIC separation mechanism     

Ion mobility–mass spectrometry: a new paradigm for proteomics

Matrix-assisted laser desorption/ionization (MALDI) coupled with ion mobility–mass spectrometry (IM–MS) provides a rapid (μs–ms) means for the two-dimensional (2D) separation of complex biological samples (e.g., peptides, oligonucleotides, glycoconjugates, lipids, etc.), elucidation of solvent-free secondary structural elements (e.g., helices, β-hairpins, random coils, etc.), rapid identification of post-translational modifications (e.g., phosphorylation, glycosylation, etc.) or ligation of small molecules, and simultaneous and comprehensive sequencing information of biopolymers. In IM–MS, protein-identification information is complemented by structural characterization data, which is difficult to obtain using conventional proteomic techniques. New avenues for enhancing the figures of merit (e.g., sensitivity, limits of detection, dynamic range, and analyte selectivity) and optimizing IM–MS experimental parameters are described in the context of deriving new information at the forefront of proteomics research.     

Searching for biomarkers of chronic obstructive pulmonary disease using proteomics: The current state

Detection of proteins which may be potential biomarkers of disorders represents a big step forward in understanding the molecular mechanisms that underlie pathological processes. In this context proteomics plays the important role of opening a path for the identification of molecular signatures that can potentially assist in early diagnosis of several clinical disturbances. Aim of this report is to provide an overview of the wide variety of proteomic strategies that have been applied to the investigation of chronic obstructive pulmonary disease (COPD), a severe disorder that causes an irreversible damage to the lungs and for which there is no cure yet. The results in this area published over the past decade show that proteomics indeed has the ability of monitoring alterations in expression profiles of proteins from fluids/tissues of patients affected by COPD and healthy controls. However, these data also suggest that proteomics, while being an attractive tool for the identification of novel pathological mediators of COPD, remains a technique mainly generated and developed in research laboratories. Great efforts dedicated to the validation of these biological signatures will result in the proof of their clinical utility.     

Novel liquid-chromatography columns for proteomics research

The enormous interest in proteomics research in recent years has inspired many developments in peptide chromatography. Different strategies have been developed to cope with the vast complexity of proteomics samples, trying to provide sufficient degree of separation to be able to exploit fully the potential of protein identification by mass spectrometry (MS). As reversed-phase liquid chromatography (RPLC) coupled to MS is still the method of choice for the analysis of protein digests, many efforts focus on the development of high-efficiency RP methods (e.g., monolithic columns and ultra-high-performance LC). This can also increase the speed and the sensitivity of the analysis of protein digests.As RPLC-MS alone is unlikely to provide sufficient resolution to unravel the composition of highly complex samples comprehensively, multidimensional methods will remain essential in proteome research. In this area, hydrophilic interaction chromatography (HILIC) seems to be a promising alternative to the traditional strong cation-exchange-based methods. Also, HILIC has found application in the analysis of post-translational modifications (e.g., phosphorylation and glycosylation).This review describes recent developments in LC methods for proteomics research, focusing on advances in column technology and the application of novel column materials. Illustrative examples show the possibilities of the new columns in proteomics research.     

Interference of the electrospray voltage on chromatographic separations using porous graphitic carbon columns

The electrospray ionization (ESI) voltage is shown to interfere with liquid chromatographic separations performed with packed porous graphitic carbon (PGC) capillary columns. This interference is ascribed to the presence of an electric field over the conductive column in the absence of an earth point between the column and the ESI emitter. The current evolved alters the chromatographic behavior of the catecholamine metabolite 3-O-methyl-DOPA significantly, as both peak splitting and a dramatic decrease in the retention time were observed. Furthermore, the response from the mass spectrometer was decreased by 33% at the same time. A related compound, tyrosine, exhibited decreased retention times but no peak splitting, whereas no shifts in the retention times (or peak splitting) were seen for the less retained dopamine and noradrenaline. When the current through the PGC column was eliminated by the use of an earth point between the column and the ESI emitter, the chromatographic behavior of the column was found to return slowly to normal after hours of equilibration with 60 : 40 (v/v) methanol-ammonium formate buffer of pH 2.9. The behavior of the PGC column with and without the earth point was found to be highly reproducible during a period of 1 month. We propose that the effect of the ESI voltage on the chromatographic behavior of the PGC column is due to associated redox reactions affecting both the PGC particles and the analytes. It is concluded that (for analytical reasons), care should be taken to ensure that no current is flowing through the chromatographic system when interfacing PGC columns, and conducting parts in general, to ESI mass spectrometry.     

Elemental mass spectrometry for quantitative proteomics

In the last decade mass-spectrometry-based proteomics has become an indispensable analytical tool for molecular biology, cellular biology and, lately, for the emerging systems biology. This review summarises the evolution and great potential of analytical methods based on elemental mass-spectrometric detection for quantitative proteomic analysis.     

Interpretation of mass spectrometry data for high-throughput proteomics

Recent developments in proteomics have revealed a bottleneck in bioinformatics: high-quality interpretation of acquired MS data. The ability to generate thousands of MS spectra per day, and the demand for this, makes manual methods inadequate for analysis and underlines the need to transfer the advanced capabilities of an expert human user into sophisticated MS interpretation algorithms. The identification rate in current high-throughput proteomics studies is not only a matter of instrumentation. We present software for high-throughput PMF identification, which enables robust and confident protein identification at higher rates. This has been achieved by automated calibration, peak rejection, and use of a meta search approach which employs various PMF search engines. The automatic calibration consists of a dynamic, spectral information-dependent algorithm, which combines various known calibration methods and iteratively establishes an optimised calibration. The peak rejection algorithm filters signals that are unrelated to the analysed protein by use of automatically generated and dataset-dependent exclusion lists. In the "meta search" several known PMF search engines are triggered and their results are merged by use of a meta score. The significance of the meta score was assessed by simulation of PMF identification with 10,000 artificial spectra resembling a data situation close to the measured dataset. By means of this simulation the meta score is linked to expectation values as a statistical measure. The presented software is part of the proteome database ProteinScape which links the information derived from MS data to other relevant proteomics data. We demonstrate the performance of the presented system with MS data from 1891 PMF spectra. As a result of automatic calibration and peak rejection the identification rate increased from 6% to 44%.Abbreviations 2-DE Two-dimensional gel electrophoresis - MALDI Matrix-assisted laser desorption ionisation - PMF Peptide mass fingerprinting - MS Mass spectrometry - TOF Time of flight     

Recent advances on multidimensional liquid chromatography–mass spectrometry for proteomics: From qualitative to quantitative analysis—A review

With the acceleration of proteome research, increasing attention has been paid to multidimensional liquid chromatography–mass spectrometry (MDLC–MS) due to its high peak capacity and separation efficiency. Recently, many efforts have been put to improve MDLC-based strategies including “top-down” and “bottom-up” to enable highly sensitive qualitative and quantitative analysis of proteins, as well as accelerate the whole analytical procedure. Integrated platforms with combination of sample pretreatment, multidimensional separations and identification were also developed to achieve high throughput and sensitive detection of proteomes, facilitating highly accurate and reproducible quantification. This review summarized the recent advances of such techniques and their applications in qualitative and quantitative analysis of proteomes.