On-line coupling of atomic spectrometry and ion chromatography with time resolved registration as a new tool for ultra trace analysis in refractory metals

Summary On-line coupling of Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES) and Ion Chromatography (IC) offers unique features for ultra trace analysis. With an on-line preconcentration procedure based on cation exchange, sub-ng/g analysis is possible in complex matrices like molybdenum and tungsten. Results match with isotope dilution mass spectrometry (IDMS) and are comparable with glow discharge mass spectrometry (GDMS). The optimal dissolution reagent for these matrices is hydrogen peroxide whose clean up to ultra high purity is possible with metal-free chromatographic equipment. All elements with cationic chemistry can be preconcentrated in acidic peroxide solutions. In this study 21 elements are measured which are detrimental for microelectronic applications. The absolute detection limits of the IC-ICP combination, with time resolved registration, are at the lower ng-level. The reproducibility is not significantly lowered by the coupling, but a ten-fold sensitivity enhancement is observed in comparison to chemically similar off-line trace-matrix-separations. Other features are simultaneous matrix corrections, use of monitors lines for method enhancement, error recognition and correction using time resolved measurement and simple calibration of the method with aqueous standards.     

Observation of sodium gel-induced protein modifications in dodecylsulfate polyacrylamide gel electrophoresis and its implications for accurate molecular weight determination of gel-separated proteins by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry (TOFMS) can potentially provide accurate molecular weight information of proteins separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Several issues related to resolution and accuracy of molecular weight measurement are investigated by using a time-lag focusing MALDI-TOF mass spectrometer. The effects of the gel components SDS, glycerol, and tris buffer on the mass spectral signals are studied systematically. Glycerol and tris buffer are shown to have little or no effect on resolution and mass accuracy, whereas SDS degrades sensitivity, resolution, and mass accuracy even at low concentrations. A simple and fast gel extraction technique is presented which is capable of detecting proteins loaded at the low-picomole level on the gel. The sample preparation procedure used in this work appears to remove most of SDS from the gel, thereby reducing the peak broadening effect caused by SDS and resulting in high resolution and accurate measurement of proteins. However, for proteins containing cysteines, the molecular ions are composed of a distribution of acrylamide-protein adducts likely formed by reaction with unpolymerized acrylamide in the gel during the gel separation process. The implications of gel-induced protein modifications on the accurate molecular weight measurement of gel-separated proteins are discussed.     

Separation and identification of photosynthetic antenna membrane proteins by high- performance liquid chromatography electrospray ionization mass spectrometry

Functional proteomics of membrane proteins is an important tool for the understanding of protein networks in biological membranes. Nevertheless, structural studies on this part of the proteome are limited. The present review attempts to cover the vast array of methods that have appeared in the last few years for separation and identification of photosynthetic proteins of thylakoid membranes present in chloroplasts, a good model for setting up analytical methods suitable for membrane proteins. The two major methods for the separation of thylakoid membrane proteins are gel electrophoresis and liquid chromatography. Isoelectric focusing in a first dimension followed by denaturing sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) in a second dimension is an effective way to resolve large numbers of soluble and peripheral membrane proteins. However, it is not applicable for isolation of native protein complexes or for the separation of highly hydrophobic membrane proteins. High-performance liquid chromatography (HPLC), on the other hand, is highly suitable for any type of membrane protein separation due to its compatibility with detergents that are necessary to keep the hydrophobic proteins in solution. With regard to the identification of the separated proteins, several methods are available, including immunological and mass spectrometric methods. Besides immunological identification, peptide mass fingerprinting, peptide fragment fingerprinting or intact molecular mass determination by electrospray ionization mass spectrometry (ESI-MS) or matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) have been shown to be very sensitive and effective. In particular, identification of proteins by their intact molecular mass is advantageous for the investigation of numerous biological problems, because it is rapid and reflects the full sequence of the protein and all its posttranslational modifications. However, intact molecular mass determinations of gel-separated membrane proteins are hampered due to the difficulties in extracting the hydrophobic proteins from the gel, whereas HPLC on-line interfaced with ESI-MS enables the rapid and accurate determination of intact molecular masses and consequently an unequivocal protein identification. This strategy can be viewed as a multidimensional separation technique distinguishing between hydrophobicity in the first dimension and between different mass-to-charge ratios in the second dimension, allowing the separation and identification even of isomeric forms.     

The role of separation science in proteomics research.

In the last few years there has been an increased effort into the separation, quantification and identification of all proteins in a cell or tissue. This is a review of the role gel electrophoresis, high performance liquid chromatography (HPLC), and capillary electrophoresis (CE) play in proteomics research. The capabilities and limitations of each separation technique have been pointed out. Instrumental strategies for the resolution of cell proteins which are based on efficient separation employing either a single high-resolution procedure or a multidimensional approach on-line or off-line, and a mass spectrometer for protein identification have been reviewed. A comparison of the advantages of multi-dimensional separations such as two-dimensional polyacrylamide gel electrophoresis, HPLC-HPLC, and HPLC-CE to the separation of cell and tissue proteins are discussed. Also, a discussion of novel approaches to protein concentration, separation, detection, and quantification is given.     

Electrospray ionization mass spectrometry of phosphopeptides isolated by on-line immobilized metal-ion affinity chromatography

Electrospray ionization mass spectrometry (ESI/MS) affords a rapid and sensitive technique for determining peptides produced by the enzymatic digestion of phosphoroteins. When coupled with on-line immobilized metal-ion affinity chromatography (IMAC), the combmation allows separation and mass spectrometric identification of phosphorylated and nonphosphorylated peptides. In this study, the feasibility and general applicability of on-line IMAC/ESI/MS is investigated by using immobilized ferric ions for selective chelation of several phosphotyrosine and phosphoserine peptides. The sensitivity and practicality of the technique for phosphoproteins are demonstrated via the analysis of 30 pmol (～0.7 μg) of bovine β-casein purified by sodium dodecylsulfate-polyacrylamide gel electrophoresis, electroblotted onto a polyvinylidene difluoride membrane, and digested in situ with trypsin. It is observed that on-line IMAC/ESI/MS suffers less from sample losses than experiments performed off-line, suggesting that the limiting factors in sensitivity for this technique are the purification procedures and sample handling rather than the IMAC and mass spectrometry. Thus, the ability to inject the tryptic digest of an electroblotted protein directly onto the column without buffer exchange and to analyze the eluent directly via on-line coupling of the IMAC column to the mass spectrometer greatly reduces sample losses incurred through sample handling and provides a convenient method for analyzing phosphopeptides at low levels.     

Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: a method for the removal of silver ions to enhance sensitivity

Mass spectrometry is a powerful technique for the identification of proteins at nanogram quantities. However, some degree of sample preparation prior to mass spectrometry is required, and silver-stained protein gel samples are most problematic. Here we report our strategy to obtain peptide mass profiles from silver-stained protein gel samples from one- or two-dimensional gels by destaining prior to enzymatic digestion. This study demonstrates that by using the destaining method, the sensitivity and quality of mass spectra is increased for matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometric analysis, permitting more proteins to be identified by peptide mass database analysis.     

Analysis of Differentially Expressed Proteins in Colorectal Cancer Using Hydroxyapatite Column and SDS-PAGE

Limitation on two dimensional (2D) gel electrophoresis technique causes some proteins to be under presented, especially the extreme acidic, basic, or membrane proteins. To overcome the limitation of 2D electrophoresis, an analysis method was developed for identification of differentially expressed proteins in normal and cancerous colonic tissues using self-pack hydroxyapatite (HA) column. Normal and cancerous colon tissues were homogenized and proteins were extracted using sodium phosphate buffer at pH 6.8. Protein concentration was determined and the proteins were loaded unto the HA column. HA column reduced the complexity of proteins mixture by fractionating the proteins according to their ionic strength. Further protein separation was accomplished by a simple and cost effective sodium dodecyl sulfate-polyacrylamide gel electrophoresis method. The protein bands were subjected to in-gel digestion and protein analysis was performed using electrospray ionization (ESI) ion trap mass spectrometer. There were 17 upregulated proteins and seven downregulated proteins detected with significant differential expression. Some of these proteins were low abundant proteins or proteins with extreme pH that were usually under presented in 2D gel analysis. We have identified brain mitochondrial carrier protein 1, T-cell surface glycoprotein CD1a, SOSS complex subunit B2, and Protein Jade 1 which were previously not detected in 2D gel analysis method.     

Chemical ionization by [NO]+ and subsequent collision-induced dissociation for the selective on-line detection of monoterpenes and linalool

Existing on-line Chemical Ionization Mass Spectrometry (CIMS) techniques for quantification of atmospheric trace gases, such as Biogenic Volatile Organic Compounds (BVOCs), suffer from difficulty in discriminating between isomeric (and more generally isobaric) compounds. Selective detection of these compounds, however, is important because they can affect atmospheric chemistry in different ways, depending on their chemical structure. In this work, Flowing Afterglow Tandem Mass Spectrometry (FATMS) was used to investigate the feasibility of the selective detection of a series of monoterpenes, an oxygenated monoterpene (linalool) and a sesquiterpene (β-caryophyllene). Ions at m/z 137 from [H(3)O](+) chemical ionization of α-pinene, linalool and β-caryophyllene have been subjected to Collision-Induced Dissociation (CID) with Ar in the collision cell of a tandem mass spectrometer at center-of-mass energies ranging between 0 and 8 eV. Similar fragmentation patterns were obtained, demonstrating that this method is not suited for the selective detection of these compounds. However, CID of the ions at m/z 136 produced via [NO](+) chemical ionization of a series of monoterpenes has revealed promising results. Some tracer-product ions for individual compounds or groups of compounds were found, which can be considered as a step forward towards selective on-line monitoring of BVOCs with CIMS techniques.     

On-line capillary separations/tandem mass spectrometry for protein digest analysis by using an ion trap storage/reflectron time-of flight mass detector

Capillary separations interfaced to tandem mass spectrometry provide a very powerful tool for the characterization of biological macromolecules such as proteins and peptides. The development of real time data-dependent data acquisition has further enhanced the capability of this method. However, the application of this technique to fast capillary separations has been limited by the relatively slow spectral acquisition speed available on scanning mass spectrometers. In this work, an ion trap storage/reflectron time-of-flight mass spectrometer (IT/reTOF-MS) has been used as an on-line tandem mass detector for capillary high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) separations of peptide mixtures including a protein digest. By taking advantage of the nonscanning property of the time-of-flight mass spectrometer, a fast spectral acquisition rate has been achieved. This fast spectral acquisition rate, combined with a new protocol that speeds up tickle voltage optimization, has provided MS/MS spectra for multiple components in a hemoglobin digest during one liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) run. Further, the IT/reTOF-MS has the speed to provide MS/MS spectra for multiple components in a CE separation of a synthetic peptide mixture within one CE/MS/MS run.     

On-line coupling of ion chromatography with ICP–AES and ICP–MS

Ion chromatography (IC) and atomic spectrometry are sometimes rivalling and sometimes ideally cooperating techniques. The cooperating applications of the on-line coupling of IC and inductively coupled plasma–atomic emission spectroscopy or –mass spectrometry span from ultra trace analysis utilizing ion exchange as preconcentration technique via speciation applications taking advantage of the unique element specific detection offered by atomic spectroscopy until classical IC applications with atomic spectrometry as a sensitive and selective detector. Characteristics of this type of hyphenated technique are the simple physical coupling, the unique sensitivity for most elements and the superior selectivity obtainable for specific applications.     

Chromatofocusing nonporous reversed-phase high-performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry of proteins from human breast cancer whole cell lysates: a novel two-dimensional liquid chromatography/mass spectrometry method

A novel two-dimensional two-column liquid chromatography/mass spectrometry (LC/MS) technique is described in this work, where chromatofocusing (CF) has been coupled to nonporous reversed-phase (NPS-RP) HPLC to separate proteins from human breast epithelial whole cell lysates. The liquid fractions from NPS-RP-HPLC are readily amenable to direct on-line analysis using electrospray ionization orthogonal acceleration time-of-flight mass spectrometry (ESI-TOFMS). A key advantage of this technique is that proteins can be 'peeled off' in the liquid phase from the CF column according to their isoelectric points (pI) in the first chromatographic separation dimension. The NPS-RP-HPLC column further separates these pI-focused fractions based upon protein hydrophobicity as the second chromatographic dimension. The third dimension involves on-line molecular weight determination using ESI-TOFMS. As a result, this method has the potential to be fully automated. In addition, a 2-D protein map of pI versus molecular weight is generated, which is analogous to a 2-D gel image. Thus, this technique may provide a means to study differential expression of proteins from whole cell lysates.     

Capillary electrophoresis fourier transform ion cyclotron resonance mass spectrometry with sustained off-resonance irradiation for the characterization of protein and peptide mixtures

A new approach to protein and peptide analysis that involves the coupling of on-line capillary electrophoresis-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry with a variation of sustained off-resonance irradiation is described. With this technique, multiple irradiation frequencies are broadcast simultaneously, which yields fragmentation of species at different mass-to-charge ratio values from the same waveform. In conjunction with capillary electrophoresis, this technique can provide sequence information from small amounts of proteins or peptides in complex mixtures. Initial results obtained from a mixture of gramicidin S (1141 u), bee venom melittin (2845 u), and equine apomyoglobin (16,951 u) are presented.     

Recent liquid chromatographic-(tandem) mass spectrometric applications in proteomics

Conventional proteomics makes use of two-dimensional gel electrophoresis followed by mass spectrometric analysis of tryptic fragments derived from in-gel digestion of proteins. Although being a very strong technique capable of separating and visualizing hundreds of proteins, 2D-gel electrophoresis has some well-documented disadvantages as well. More recently, liquid chromatographic-(tandem) mass spectrometric techniques have been developed to overcome some of the shortcomings of 2D-gel electrophoresis. In this review we have described several recent applications of liquid chromatography-(tandem) mass spectrometry in the field of proteomics and especially in the field of membrane proteomics, quantitative proteomics and in the analysis of post-translational modifications.     

Two-dimensional strong cation exchange/porous layer open tubular/mass spectrometry for ultratrace proteomic analysis using a 10 microm id poly(styrene- divinylbenzene) porous layer open tubular column with an on-line triphasic trapping column

This study expands the capabilities for ultratrace proteomic analysis of our previous work by incorporating on-line sample desalting using a triphasic (RP/strong cation exchange (SCX)/micro-SPE) trapping column connected to a 3.2 m x 10 microm id poly(styrene-divinylbenzene) (PS-DVB) porous layer open tubular (PLOT) column. To minimize extra sample handling steps, C18 RP packing was incorporated in the capillary tubing upstream of the SCX column for the on-line desalting. For the micro-SPE column, a 50 microm id PS-DVB monolithic column was positioned downstream of the SCX column. High-performance separation was achieved on the PLOT column at a mobile phase flow rate of 20 nL/min. The sensitivity and high resolution capability of the new multidimensional platform was evaluated using an in-gel tryptic digested sample of a cervical cancer (SiHa) cell line. For the injected amount of 1200 cells ( approximately 500 ng), over 2700 peptides covering greater than 850 unique proteins were identified from the triphasic SCX/PLOT/MS analysis of a single SDS gel section (>40 kDa). The 2-D LC/MS platform demonstrated good separation performance, such that more than 85% of the identified peptides were detected from only one salt fraction. In a triplicate analysis of the above >40 kDa gel section, 4497 peptides and 1209 unique proteins were identified when applying stringent filtering criteria, with a false-positive rate of 2.4%. When all three SDS-PAGE gel sections of the lysed SiHa cells were analyzed, 5047 peptides and 1857 unique proteins (false-positive rate 1.8%), including cancer-related proteins such as MAP kinases, were identified.     

Performance of capillary gel electrophoretic analysis of oligonucleotides coupled on-line with electrospray mass spectrometry

Synthetic oligonucleotides (ODNs) are routinely analyzed using capillary gel electrophoresis (CGE) for size-sieving based separations as well as electrospray mass spectrometry (ESI-MS) for identification. On-line coupling of these methods is therefore desired in order to combine the analytical capabilities provided by both methods. Performance of on-line CGE-ESI-MS systems is influenced by various parameters, and choice of optimal conditions is crucial for successful coupling experiments. In this study, we explore characteristics of the on-line coupled CGE-ESI-MS system for ODN analysis. Effects of CGE buffer concentration, capillary length, separation and orifice voltage on CGE separation and MS detection of a phosphodiester ODN mixture were examined. Attention was paid to the influence of the interface, such as geometry of capillary alignment, sheath liquid flow-rate and sheath liquid composition on performance of the system.     

On-line coupling of ion chromatography and atomic spectrometry for ultra trace analysis in high purity molybdenum- and tungsten-silicide

The well-established technique of on-line coupling ion chromatography and atomic spectrometry for ultra trace analysis in high purity molybdenum and tungsten is extended to include the silicides MoSi(x) and WSi(x). An additionally included matrix elimination step allows an almost interference-free trace analysis in the silicide matrices. Reproducibility and accuracy of the on-line method were checked by comparison with several other methods, such as isotope dilution, radiochemical neutron activation analysis, direct determination by atomic absorption analysis and not at least with glow discharge mass spectrometry. The results show the high potential of the on-line method for reaching detection limits in the pg g(-1) range, but they show also remaining problems with contamination and system calibration.     

Applications of Tandem Mass Spectrometry (MS/MS) in Protein Analysis for Biomedical Research

Mass Spectrometry (MS) allows the analysis of proteins and peptides through a variety of methods, such as Electrospray Ionization-Mass Spectrometry (ESI-MS) or Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry (MALDI-MS). These methods allow identification of the mass of a protein or a peptide as intact molecules or the identification of a protein through peptide-mass fingerprinting generated upon enzymatic digestion. Tandem mass spectrometry (MS/MS) allows the fragmentation of proteins and peptides to determine the amino acid sequence of proteins (top-down and middle-down proteomics) and peptides (bottom-up proteomics). Furthermore, tandem mass spectrometry also allows the identification of post-translational modifications (PTMs) of proteins and peptides. Here, we discuss the application of MS/MS in biomedical research, indicating specific examples for the identification of proteins or peptides and their PTMs as relevant biomarkers for diagnostic and therapy.     

A robust new strategy for high-molecular-weight proteome research: A 2-hydroxyethyl agarose/polyacrylamide gel enhanced separation and ZnO-PMMA nanobeads assisted identification

A new mass spectrometry based analysis strategy has been established here for high-molecular-weight (HMW) proteome research. First, a 2-hydroxyethyl agarose/polyacrylamide (HEAG/PAM) electrophoresis gel was designed for the first time to realize an easy-handling separation method with high spatial resolution for HMW proteins, good reproducibility and mass spectrometry-compatible sliver staining. Second, ZnO-polymethyl methacrylate (ZnO-PMMA) nanobeads were applied here for enriching and desalting the peptides from the HMW proteins. Third, the peptides were analyzed by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) with the presence of the ZnO-PMMA nanobeads, and their MS signals were enhanced markedly. The success rate of identification for HMW proteins was significantly increased due to high enriching efficiency and salt tolerance capability as well as signal enhancing capability of the ZnO-PMMA nanobeads. We believe that this analysis strategy will inspire and accelerate the HMW proteome studies.     

Novel procedure for the identification of proteins by mass fingerprinting combining two-dimensional electrophoresis with fluorescent SYPRO red staining

The fluorescent sensitive SYPRO Red dye was successfully employed to stain proteins in two-dimensional gels for protein identification by peptide mass fingerprinting. Proteins which are not chemically modified during the SYPRO Red staining process are well digested enzymatically in the gel and hence the resulting peptides can be efficiently eluted and analysed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). A SYPRO Red two-dimensional gel of a complex protein extract from Candida albicans was analysed by MALDI-TOF MS. The validity of SYPRO Red staining was demonstrated by identifying, via peptide mass fingerprinting, 10 different C. albicans proteins from a total of 31 selected protein spots. The peptide mass signal intensity, the number of matched peptides and the percentage of coverage of protein sequences from SYPRO Red-stained proteins were similar to or greater than those obtained in parallel with the modified silver protein gel staining. This work demonstrates that fluorescent SYPRO Red staining is compatible with the identification of proteins separated on polyacrylamide gel and that it can be used as an alternative to silver staining. As far as we know, this is the first report in which C. albicans proteins separated using 2-D gels have been identified by peptide mass fingerprinting. The improved technique described here should be very useful for carrying out proteomic studies.     

Micro-high-performance liquid chromatography platform for the depletion of high-abundance proteins and subsequent on-line concentration/capturing of medium and low-abundance proteins from serum. Application to profiling of protein expression in healthy and osteoarthritis sera by 2-D gel electrophoresis

In this investigation, an integrated microcolumn-based fluidic platform for the simultaneous depletion of high-abundance proteins and the subsequent on-line concentration/capturing of medium- and low-abundance proteins from human serum has been introduced. The platform consists of on-line coupling of tandem affinity micorcolumns to an RP microcolumn to achieve first the depletion of high-abundance proteins by the tandem affinity microcolumns followed by the concentration and capturing of medium- and low-abundance proteins by the RP microcolumn. The tandem affinity microcolumns are based on macroporous monoliths characterized by their relatively high permeability in pressure-driven flow while the RP microcolumn is packed with polymeric particles with an average particle diameter of 20 microm giving rise to a very little back pressure, thus allowing fast flow velocity across the coupled columns format and consequently short processing time of serum samples prior to analysis by 2-DE. The microcolumn-based fluidic platform was applied to serum samples from osteoarthritis (OA) donors before and after soy protein (SP) supplementation, and from healthy donors, and the resulting depleted serum samples from high-abundance proteins were profiled for protein expression by 2-DE. In general, the protein expression was lower in serum of the same OA patient after soy treatment than before soy treatment. Several proteins were down-regulated after soy treatment with transthyretin being the most affected by the SP supplementation. In addition, with respect to serum from healthy donors, the sera from OA patients showed difference in proteins expression.