Automated nanoflow liquid chromatography-tandem mass spectrometry for a differential display proteomic study on Xenopus laevis neuroendocrine cells

Many proteomic projects based on a comparison of protein profiles displayed on two-dimensional polyacrylamide gel electrophoresis rely on the identification of these proteins using peptide mass fingerprinting on a matrix-assisted laser desorption/ionization mass spectrometer after tryptic digestion. However, this approach is limited to an organism of which genomic information is largely available, i.e. when the total genome sequence is known. For other organisms, mass spectrometric sequence analysis is necessary for protein identification. We established a nano-LC-MS-MS system based on a quadrupole time-of-flight mass spectrometer, which allows automated sequence analysis of tryptic digestion mixtures from single gel spots. This system is applied in a differential-display proteomic study to identify differentially expressed proteins in the neuroendocrine cells of the neurointermediate pituitary of black- and white-background adapted Xenopus laevis.     

CHASE, a charge-assisted sequencing algorithm for automated homology-based protein identifications with matrix-assisted laser desorption/ionization time-of-flight post-source decay fragmentation data

We describe CHASE, a novel algorithm for automated de novo sequencing based on the mass spectrometric (MS) fragmentation analysis of tryptic peptides. This algorithm is used for protein identification from sequence similarity criteria and consists of four steps: (1) derivatization of tryptic peptides at the N-terminus with a negatively charged reagent; (2) post-source decay (PSD) fragmentation analysis of peptides; (3) interpretation of the mass peaks with the CHASE algorithm and reconstruction of the amino acid sequence; (4) transfer of these data to software for protein identifications based on sequence homology (Basic Local Alignment Search Tool, BLAST). This procedure deduced the correct amino acid sequence of tryptic peptide samples and also was able to deduce the correct sequence from difficult mass patterns and identify the amino acid sequence. This allows complete automation of the process starting from MS fragmentation of complex peptide mixtures at low concentration (e.g. from silver-stained gel bands) to identification of the protein. We also show that if PSD data are collected in a single spectrum (instead of the segmented mode offered by conventional matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) instrumentation), the complete workflow from MS-PSD data acquisition to similarity-based identification can be completely automated. This strategy may be applied to proteomic studies for protein identification based on automated de novo sequencing instead of MS or tandem MS patterns. We describe the Charge Assisted Sequencing Engine (CHASE) algorithm, the working protocol, the performance of the algorithm on spectra from MALDI-TOFMS and the data comparison between a TOF and a TOF-TOF instrument.     

The extent and effects of peptide sequence scrambling via formation of macrocyclic <Emphasis Type="Italic">b</Emphasis> ions in model proteins

The extent and effects of sequence scrambling in peptide ions during tandem mass spectrometry (MS/MS) have been examined using tryptic peptides from model proteins. Sequencescrambled b ions appeared in about 35% of 43 tryptic peptides examined under MS/MS conditions. In general, these ions had relatively low abundances with averages of 8% and 16%, depending on the instrumentation used. A few tryptic peptides gave abundant scrambled b ions in MS/MS. However, peptide and protein identifications under proteomic conditions with Mascot were not affected, even for these peptides wherein scrambling was prominent. From the 43 tryptic peptides that have been investigated, the conclusion is that sequence scrambling is unlikely to impact negatively on the accuracy of automated peptide and protein identifications in proteomics.     

Rapid and sensitive drug metabolism studies by SU-8 microchip capillary electrophoresis-electrospray ionization mass spectrometry

Monolithically integrated, polymer (SU-8) microchips comprising an electrophoretic separation unit, a sheath flow interface, and an electrospray ionization (ESI) emitter were developed to improve the speed and throughput of metabolism research. Validation of the microchip method was performed using bufuralol 1-hydroxylation via CYP450 enzymes as the model reaction. The metabolite, 1-hydroxybufuralol, was easily separated from the substrate (R(s)=0.5) with very good detection sensitivity (LOD=9.3nM), linearity (range: 50-500nM, r(2)=0.9997), and repeatability (RSD(Area)=10.3%, RSD(Migrationtime)=2.5% at 80nM concentration without internal standard). The kinetic parameters of bufuralol 1-hydroxylation determined by the microchip capillary electrophoresis (CE)-ESI/mass spectrometry (MS) method, were comparable to the values presented in literature as well as to the values determined by in-house liquid chromatography (LC)-UV. In addition to enzyme kinetics, metabolic profiling was demonstrated using authentic urine samples from healthy volunteers after intake of either tramadol or paracetamol. As a result, six metabolites of tramadol and four metabolites of paracetamol, including both phase I oxidation products and phase II conjugation products, were detected and separated from each other within 30-35s. Before analysis, the urine samples were pre-treated with on-chip, on-line liquid-phase microextraction (LPME) and the results were compared to those obtained from urine samples pre-treated with conventional C18 solid-phase extraction (SPE, off-chip cartridges). On the basis of our results, the SU-8 CE-ESI/MS microchips incorporating on-chip sample pre-treatment, injection, separation, and ESI/MS detection were proven as efficient and versatile tools for drug metabolism research.     

Microchip capillary electrophoresis–electrospray ionization–mass spectrometry of intact proteins using uncoated Ormocomp microchips

We present rapid (<5 min) and efficient intact protein analysis by mass spectrometry (MS) using fully microfabricated and monolithically integrated capillary electrophoresis–electrospray ionization (CE–ESI) microchips. The microchips are fabricated fully of commercial inorganic–organic hybrid material, Ormocomp, by UV-embossing and adhesive Ormocomp–Ormocomp bonding (CE microchannels). A sheath-flow ESI interface is monolithically integrated with the UV-embossed separation channels by cutting a rectangular emitter tip in the end with a dicing saw. As a result, electrospray was produced from the corner of chip with good reproducibility between parallel tips (stability within 3.8–9.2% RSD). Thanks to its inherent biocompatibility and stable (negative) surface charge, Ormocomp microchips enable efficient intact protein analysis with up to ∼10⁴ theoretical separation plates per meter without any chemical or physical surface modification before analysis. The same microchip setup is also feasible for rapid peptide sequencing and mass fingerprinting and shows excellent migration time repeatability from run to run for both peptides (5.6–5.9% RSD, n = 4) and intact proteins (1.3–7.5% RSD, n = 3). Thus, the Ormocomp microchips provide a versatile new tool for MS-based proteomics. Particularly, the feasibility of the Ormocomp chips for rapid analysis of intact proteins with such a simple setup is a valuable increment to the current technology.     

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.     

Protein identification from two-dimensional gel electrophoresis by connecting ZipTip with a home-assembled nanoflow system

Mass spectrometry (MS) is a powerful technique for protein identification in proteomic research. Two-dimensional gel electrophoresis (2-DE) combined with MS is a significant method for protein separation and identification. For protein identification, peptide sequencing is usually carried out by an effective but expensive nano-flow liquid chromatographic system combined to tandem mass spectrometry (MS/MS). However, protein identification based on such method is time-consuming, and contamination may occur as a result of column overloading. In this study, we establish an alternative nanoscale system for protein identification using MS/MS. The system consists of several devices that can be purchased from commercial sources and can be connected to an electrospray ionization quadrupole-time of flight (ESI-Q-TOF) MS in order to analyze proteins from 2D gels. This inexpensive strategy provides an attractive alternative method for rapid identification of proteins using a nanospray source. In addition, the device is disposable so that contamination is avoided. It is shown that peptide sequencing based on this device using ESI-Q-TOF MS is accomplished within 10 min.     

Novel polymer composite to eliminate background matrix ions in matrix assisted laser desorption/ionization-mass spectrometry

Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) is rarely used for the analysis of small molecules (< 700 Da) because the low m/z signal is overwhelmed by a high background of matrix ions. We have developed a solution to this problem that employs a novel polymer composite which is formed by covalently cross-linking alpha-cyano-4-hydroxycinnamic acid (HCCA) to SU-8 photoresist via cationic photo-polymerization. Since the HCCA molecules are immobilized, background noise resulting from the matrix ions is significantly reduced or eliminated. Moreover, owing to the hydrophobic surface of the polymer film, the sample spots shrink during solvent evaporation and thus the analytes can be concentrated. As a result, this polymer composite improves detection sensitivity and extends the analyzable species to the low-mass region. The covalent incorporation of HCCA with SU-8 was validated with reflectance FTIR spectroscopy, and the polymer surface was characterized with scanning electron microscopy (SEM). Using MRFA, a small peptide as a standard, 8 mg of HCCA per mL of SU-8 photoresist was found to yield the highest sensitivity and the lowest background noise. Analytes such as peptides or small organic molecules were further examined on this composite surface and no analyte degradation was observed. In a trial of peptide mass fingerprinting of cytochrome c on the composite substrate, the inclusion of low m/z tryptic peptides in the database search dramatically improved the protein identification probability score.     

Integrated microfabricated systems including a purification module and an on-chip nano electrospray ionization interface for biological analysis

We report here on an integrated microfabricated device dedicated to the preparation of biological samples prior to their on-line analysis by electrospray ionization-mass spectrometry (ESI-MS). This microfluidic device is fabricated using the negative photoresist SU-8 by microtechnology techniques. The device includes a chromatographic module plus an ESI interface for MS. The chromatographic module is dedicated to sample purification and is based on a polymer monolithic phase which includes hydrophobic moieties. The ESI interface is integrated onto the chip and is based on a capillary slot. We present here the integration of these different modules onto a single system that is fabricated via a SU-8-based microtechnology route. We present also their testing for the purification of peptide samples. This started with a partial integration step with the combination of at least two of the modules (microsystem + monolith; microsystem + nib) and their test before the fabrication and testing of fully integrated microsystems.     

Reliable detection of milk allergens in food using a high-resolution, stand-alone mass spectrometer

Reliable methods are needed for detection of allergenic milk proteins in complex food matrixes. The feasibility of an LC/high-resolution MS method for the analysis of milk proteins in a thermally processed model food (incurred cookies) and in white wine spiked, respectively, with milk powder and caseinate is described. Detection of milk proteins was based on the identification of unique peptides in the tryptic digests of cookie/wine extracts using an RP-HPLC separation coupled to an Exactive nonhybrid mass spectrometer using Orbitrap technology. The extremely high mass accuracy and resolution provided by the Orbitrap analyzer allowed a fast preliminary identification of four previously proposed peptide markers of caseins using only accurate values of the m/z of their ions. No interference was observed, despite the complexity of the analyzed matrixes. Moreover, the availability of a high- energy, collisionally activated dissociation cell integrated in the mass spectrometer enabled acquisition of peptide MS/MS-like spectra through post-source fragmentation. Confirmation of peptide marker identity could then be achieved by a comparison between experimental and predicted product ions. The described method shows the great potential of Orbitrap MS as a reliable technique in the field of protein allergen detection once the peptide markers are identified.     

Identification of low molecular weight proteins isolated by 2-D liquid separations

Proteins with molecular mass (M(r)) <20 kDa are often poorly separated in 2-D sodium dodecyl sulfate polyacrylamide gel electrophoresis. In addition, low-M(r) proteins may not be readily identified using peptide mass fingerprinting (PMF) owing to the small number of peptides generated in tryptic digestion. In this work, we used a 2-D liquid separation method based on chromatofocusing and non-porous silica reversed-phase high-performance liquid chromatography to purify proteins for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOFMS) analysis and protein identification. Several proteins were identified using the PMF method where the result was supported using an accurate M(r) value obtained from electrospray ionization TOFMS. However, many proteins were not identified owing to an insufficient number of peptides observed in the MALDI-TOF experiments. The small number of peptides detected in MALDI-TOFMS can result from internal fragmentation, the few arginines in its sequence and incomplete tryptic digestion. MALDI-QTOFMS/MS can be used to identify many of these proteins. The accurate experimental M(r) and pI confirm identification and aid in identifying post-translational modifications such as truncations and acetylations. In some cases, high-quality MS/MS data obtained from the MALDI-QTOF spectrometer overcome preferential cleavages and result in protein identification.     

Epitope mapping of monoclonal antibodies by mass spectrometry: Identification of protein antigens in complex biological systems

We describe the application of immunoaffinity extraction and mass spectrometry to the analysis of Ty1 Gag protein in lysates of Saccharomyces cerevisiae. A magnetic bead-conjugated monoclonal antibody was used to achieve selective extraction, the specificity of which was established by matrix-assisted laser desorption/ionization mass spectrometric (MS) analysis of an extract of the lysate of cells overexpressing the Ty1 Gag protein. MS analysis of similar extracts of lysates following tryptic hydrolysis confirmed selective extraction of the epitope-containing peptide fragment. Sufficient sensitivity was achieved to allow the application of this approach to the analysis of lysates of wild-type cells. Furthermore, the sequence of the epitope-containing peptide was confirmed by electrospray-tandem MS. To our knowledge, this constitutes the first report of the application of immunoaffinity extraction and tandem MS analysis to the characterization of an antigen recovered from a complex cellular system.     

Towards a human repertoire of monocytic lysosomal proteins

The lysosomal compartment of human monocytic cells has never been investigated by a proteomic approach. By a combination of one-dimensional (1-D) and two-dimensional (2-D) gel electrophoresis, protein identification by N-terminal sequencing, matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) peptide mass fingerprinting and tandem mass spectrometry (MS/MS) peptide sequence analysis, we initiated an exhaustive study of the human lyososomal proteome, which aims at establishing a 2-D reference map of human soluble lyososomal proteins. Human monocytic U937 cells were induced to secrete lysosomal soluble hydrolases by addition of NH4Cl in the culture medium. Since lysosomal soluble proteins are characterized by the presence of mannose-6-phosphate, they were purified on an affinity support bearing mannose-6-phosphate receptor. Analysis of the purified fraction led to the preliminary identification of fifteen proteins, among which twelve are well-known lysosomal hydrolases, one is assumed to be lysosomal on the basis of sequence homology to cysteine proteinases of the papain family, and two (leukocystatin and the human cellular repressor of E1A-stimulated genes) are described here for the first time as mannose-6-phosphate-containing proteins.     

蛋白质酶解混合物的动态修饰毛细管电泳-质谱联用分析

利用羟丙基纤维素溶液动态涂层技术修饰毛细管管壁,改善了分离效率.在不影响质谱检测的条件下,将动态涂层毛细管电泳与质谱检测联用,有效地提高了对蛋白质的鉴定能力.将该技术应用于对复杂蛋白质样品的酶解产物的分析鉴定,结果令人满意.     

Chemically-assisted fragmentation combined with multi-dimensional liquid chromatography and matrix-assisted laser desorption/ionization post source decay, matrix-assisted laser desorption/ionization tandem time-of flight or matrix-assisted laser desorption/ionization tandem mass spectrometry for improved sequencing of tryptic peptides

Derivatization of tryptic peptides using an Ettan CAF matrix-assisted laser desorption/ionization (MALDI) sequencing kit in combination with MALDI-post source decay (PSD) is a fast, accurate and convenient way to obtain de novo or confirmative peptide sequencing data. CAF (chemically assisted fragmentation) is based on solid-phase derivatization using a new class of water stable sulfonation agents, which strongly improves PSD analysis and simplifies the interpretation of acquired spectra. The derivatization is performed on solid supports, ZipTip(microC18, limiting the maximum peptide amount to 5 microg. By performing the derivatization in solution enabled the labeling of tryptic peptides derived from 100 microg of protein. To increase the number of peptides that could be sequenced, derivatized peptides were purified using multidimensional liquid chromatography (MDLC) prior to MALDI sequencing. Following the first dimension strong cation exchange (SCX) chromatography step, modified peptides were separated using reversed-phase chromatography (RPC). During the SCX clean up step, positively charged peptides are retained on the column while properly CAF-derivatized peptides (uncharged) are not. A moderately complex tryptic digest, prepared from six different proteins of equimolar amounts, was CAF-derivatized and purified by MDLC. Fractions from the second dimension nano RPC step were automatically sampled and on-line dispensed to MALDI sample plates and analyzed using MALDI mass spectrometry fragmentation techniques. All proteins in the derivatized protein mixture digest were readily identified using MALDI-PSD or MALDI tandem mass spectrometry (MS/MS). More than 40 peptides were unambiguously sequenced, representing a seven-fold increase in the number of sequenced peptides in comparison to when the CAF-derivatized protein mix digest was analyzed directly (no MDLC-separation) using MALDI-PSD. In conclusion, MDLC purification of CAF-derivatized peptides significantly increases the success rate for de novo and confirmative sequencing using various MALDI fragmentation techniques. This new approach is not only applicable to single protein digests but also to more complex digests and could, thus, be an alternative to electrospray ionization MS/MS for peptide sequencing.     

Identification of proteins separated by one-dimensional sodium dodecyl sulfate/polyacrylamide gel electrophoresis with matrix-assisted laser desorption/ionization ion trap mass spectrometry; comparison with matrix-assisted laser desorption/ionization time-of-flight mass fingerprinting

Digests from ten gel bands containing low abundance proteins were analyzed by both matrix-assisted laser desorption/ionization ion trap (MALDI-IT) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) methods. MALDI-TOF techniques were able to identify only one protein from all 10 gel bands, while MALDI-IT identified eight proteins from the same 10 bands. The ability to perform MS/MS experiments with a MALDI-IT instrument leads to protein identifications based on both peptide molecular mass and sequence information, and is much less prone to errors and uncertainties introduced by peptide fingerprinting methodologies in which protein identification is based on peptide molecular masses alone.     

Nanodiamond MALDI support for enhancing the credibility of identifying proteins

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a standard analytical tool for protein identification and peptide sequencing. High sensitivity and resolution are two critical parameters for recording good peptide mass fingerprinting (PMF) of low abundance proteins. Here, we report a novel nanodiamond (ND) (normal size 3–10 nm) support for MALDI-MS target, over which -cyano-4-hydrocinnamic acid (CCA) crystallizes evenly. Good reproducibility of relative peak intensity (R.S.D. less than 11.8%) among sample spot (from ring to center) is achieved on ND support. Therefore, the search for “hot spots” during the analysis is not necessary, which is supporting for the automatic acquisition of data. Due to high absorbability of energy from the laser, the ND support improves ionization efficiency of samples. In general, the sensitivity of MS obtained on ND support can be enhanced three to four times compared to the conventional MALDI sample preparation technique. Sensitivity obtained on ND support ranges from 62.5 amol of Arg-vasopressin standard peptide to 1.0 fmol of myoglobin tryptic peptide mixture. Reduced spot size and increased sensitivity in MALDI-MS are also accomplished by ND support. With spot size reduced, the signal intensity of cytochrome c (Cyt c) tryptic peptide obtained on ND support is at least seven times greater than it acquired on stainless steel. And ND support has been found better tolerance for salt (up to 500 mM NaCl) to MALDI-MS analysis. All these properties make ND support a valuable tool for MALDI-MS identification of proteins.     

基于毛细管电泳技术的高灵敏度蛋白质组学技术发展

近年来,蛋白质组学技术在样品前处理、分离技术和质谱检测技术方面获得了快速发展,已经可以实现在几小时内对上万种蛋白的同时定性和定量分析。然而,目前的主流蛋白质组学技术仍无法满足极微量生物样品,尤其是单细胞样品的组学分析需求。毛细管电泳分离技术具有峰宽窄、柱效高、样品用量少等优势,是与高分辨质谱在线联用的理想选择之一。该文评述了集成化和在线样品前处理以及主流的纳升液相色谱-质谱联用系统在高灵敏度蛋白质组学分析领域的发展现状和挑战,认为该领域的重要技术挑战之一在于目前的纳升液相色谱分离已经无法完全匹配现代高分辨质谱超过40 Hz的超高扫描速度,从而导致质谱使用效率的降低。针对上述技术挑战,该文重点探讨了毛细管电泳-质谱联用技术的独特技术优势和潜在发展机遇,主要包括：（1）面向微量酶解多肽样品的高柱效毛细管电泳分离。通过采用毛细管电色谱可以进一步改善毛细管电泳柱容量不足的局限;（2）面向高灵敏度分析的无鞘液/鞘液接口开发;（3）高效毛细管电泳分离与高扫描速度质谱检测的协同化使用。总之,我们预期毛细管电泳-质谱联用技术的进一步发展有望在针对单细胞等超微量生物学样品的蛋白质组学分析中获得更广泛的应用。     

Interrogation of MS/MS search data with an pI Filter algorithm to increase protein identification success

In high-throughput proteomics, the bottom-up approach has become a widely used method for the identification of proteins that is based on tryptic peptide MS/MS analysis. Separation methodologies that use IEF of tryptic peptides have recently been introduced and provide an extra dimension of peptide separation. In addition to its great fractionation capability, tryptic peptide prefractionation by IEF can also increase the protein identification success. The pI information of the peptide gained can be successfully used in a post-database search filtering step. We introduce a filtering algorithm that is based on the comparison of the experimental and theoretical pI's to validate peptide identifications by MS/MS data search engines.