Comparison of different separation technologies for proteome analyses: isoform resolution as a prerequisite for the definition of protein biomarkers on the level of posttranslational modifications

In this article we evaluate methods used to reveal the molecular complexity, which is generated in biological samples by posttranslational modifications (PTM) of proteins. We show how distinct molecular differences on the level of phosphorylation sites in a single protein (ovalbumin) can be resolved with different success using 1D and 2D gel-electrophoresis and reversed-phase liquid chromatography (LC) with monolithic polystyrol-divinylbenzol (PS-DVB) columns for protein separation, and matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) for protein identification. Phosphorylation site analysis was performed using enzymatic dephosphorylation in combination with differential peptide mass mapping. Liquid chromatography-MALDI-TOF MS coupling with subsequent on-target tryptic protein digestion turned out to be the fastest method tested but yielded low resolution for the analysis of PTM, whereas 2D gel-electrophoresis, due to its unique capability of resolving highly complex isoform pattern, turned out to be the most suitable method for this purpose. The evaluated methods complement one another and in connection with efficient technologies for differential and quantitative analysis, these approaches have the potential to reveal novel molecular details of protein biomarkers.     

Study of posttranslational non-enzymatic modifications of collagen using capillary electrophoresis/mass spectrometry and high performance liquid chromatography/mass spectrometry

The depository effects that occur in slowly metabolized proteins (typically glycation) are very difficult to assess, owing to their extremely low concentration in the protein matrix. Collagen accumulates reactive metabolites through reactions that are not regulated by enzymes. A typical example of these non-enzymatic changes is glycation (the Maillard reaction, the formation of advanced glycation end products), resulting from the reaction of the oxo-group of sugars with the epsilon-amino group of lysine and arginine. Collagen samples (type I) as a test protein were incubated separately with glucose, ribose and malondialdehyde. Collagen was fragmented with cyanogen bromide and then digested with trypsin. This peptide digest was separated by CE, CE-MS/MS, and HPLC-MS/MS. An ion trap MS was used and MS conditions were optimized for both methods. These on-line CE-MS/MS and HPLC-MS/MS couplings made it possible to discover specific modifications such as (N(epsilon)-(carboxymethyl)-lysine) in the precise location in the structure of collagen corresponding to posttranslational non-enzymatic modifications. A new CE-MS/MS technique for peptide analysis was developed, and applied in the identification of posttranslational modifications in slowly metabolized test proteins.     

Novel staining-free proteomic method for simultaneous identification of proteins and determination of their pI values by using low-molecular-mass pI markers

A new proteomic staining-free method for simultaneous identification of proteins and determination of their pI values by using low-molecular-mass pI markers is described. It is based on separation of proteins in gels by IEF in combination with mass spectrometric analysis of both peptides derived by in-gel digestion and low-molecular-mass pI markers extracted form the same piece excised from the gel. In this method, the pI markers are mixed with a protein mixture (a commercial malted barley protein extract) deposited on a gel and separated in a pH gradient. Color pI markers enable supervision of progress of focusing process. Several separated bands of the pI markers (including separated proteins) were excised and the pI markers were eluted from each gel piece by water/ethanol and identified by MALDI-TOF/TOF MS. The remaining carrier ampholytes were then washed out from gel pieces and proteins were in-gel digested with trypsin or chymotrypsin. Obtained peptides were measured by MALDI-TOF/TOF MS and proteins were identified via protein database search. This procedure allows omitting time-consuming protein staining and destaining procedures, which shortens the analysis time. For comparison, other IEF gels were stained with CBB R 250 and proteins in the gel bands were identified. Similarity of the results confirmed that our approach can give information about the correct pI values of particular proteins in complex samples at significantly shorter analysis times. This method can be very useful for identification of proteins and their post-translational modifications in prefractioned samples, where post-translational modifications (e.g., glycation) are frequent.     

Application of proteomics to hordein screening in the malting process

This study was undertaken to investigate the effect of the malting process on hordein composition. For this purpose, combination of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and the method of isotopic peptides labeling iTRAQ was used. Barley proteins are essential components determining the quality of both malt and beer. Since hordeins represent the most abundant proteins accounting for about 40-50% of total protein fraction of mature barley grain, our research was focused on them. In this respect, the proteins of interest were extracted from milled samples of barley grain, germinated barley grain (samples collected at different time intervals), green malt and malt, respectively. Particular hordein extracts were firstly fractionated via SDS- PAGE, which was used as a relatively rapid and reliable technique providing information about hordein profile of analyzed samples. Then, separated proteins were in-gel digested and resulting peptides were measured by mass spectrometry. In addition, the chosen proteins, after in-gel digestion, were subjected to the iTRAQ method and the screening of proteins during malting process was evaluated. Our results have revealed that most of the hordein components present in the barley grain can be found in all stages of the malting process as well as in the final malt. The amount of hordeins decreases during the malting process; in the case of C hordein, the protein decrease is approximately 65%. On the other hand, significant degradation of D hordein was detected. The suggested procedure can be used to follow the development of the hordein profile during germination, which is of great technological importance in beer production.     

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.     

Use of monolithic supports in proteomics technology

An overview on the utilization of monoliths in proteomics technology will be given. Both silica- and polymer-based monoliths have broad use for microseparation of tryptic peptides in reversed-phase (RP) mode before identification by mass spectrometry (MS) or by MS/MS. For two-dimensional (2D) LC separation of peptides before MS or MS/MS analysis, a combination of ion-exchange, usually cation-exchange (CEX) chromatography with RP chromatography on monolithic supports can be employed. Immobilized metal ion affinity chromatography monoliths with immobilized Fe3+-ions are used for the isolation of phosphopeptides. Monoliths with immobilized affinity ligands are usually applied to the rapid separation of proteins and peptides. Miniaturized reactors with immobilized proteolytic enzymes are utilized for rapid on- or offline digestion of isolated proteins or protein mixtures prior to identification by LC-MS/MS. Monoliths also have broad potential for application in sample preparation, prior to further proteomic analyses. Monolithic supports with large pore sizes can be exploited for the isolation of nanoparticles, such as cells, organelles, viruses and protein aggregates. The potential for further adoption of monolithic supports in protein separation and enrichment of low abundance proteins prior to proteolytic digestion and final LC-MS/MS protein identification will be discussed.     

Microwave-assisted acid hydrolysis of proteins combined with liquid chromatography MALDI MS/MS for protein identification

Simple and efficient digestion of proteins, particularly hydrophobic membrane proteins, is of significance for comprehensive proteome analysis using the bottom-up approach. We report a microwave-assisted acid hydrolysis (MAAH) method for rapid protein degradation for peptide mass mapping and tandem mass spectrometric analysis of peptides for protein identification. It uses 25% trifluoroacetic acid (TFA) aqueous solution to dissolve or suspend proteins, followed by microwave irradiation for 10 min. This detergent-free method generates peptide mixtures that can be directly analyzed by liquid chromatography (LC) matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) without the need of extensive sample cleanup. LC-MALDI MS/MS analysis of the hydrolysate from 5 microg of a model transmembrane protein, bacteriorhodopsin, resulted in almost complete sequence coverage by the peptides detected, including the identification of two posttranslational modification sites. Cleavage of peptide bonds inside all seven transmembrane domains took place, generating peptides of sizes amenable to MS/MS to determine possible sequence errors or modifications within these domains. Cleavage specificity, such as glycine residue cleavage, was observed. Terminal peptides were found to be present in relatively high abundance in the hydrolysate, particularly when low concentrations of proteins were used for MAAH. It was shown that these peptides could still be detected from MAAH of bacteriorhodopsin at a protein concentration of 1 ng/microl or 37 fmol/microl. To evaluate the general applicability of this method, it was applied to identify proteins from a membrane protein enriched fraction of cell lysates of human breast cancer cell line MCF7. With one-dimensional LC-MALDI MS/MS, a total of 119 proteins, including 41 membrane-associated or membrane proteins containing one to 12 transmembrane domains, were identified by MS/MS database searching based on matches of at least two peptides to a protein.     

Mass spectrometric analysis of porcine rhodopsin

Rhodopsin is the dim light photosensitive pigment of animals. In this work, we undertook to study the structure of rhodopsin from swine and compare it with bovine and rat rhodopsin. Porcine rhodopsin was analyzed using methodology developed previously for mass spectrometric analysis of integral membrane proteins. Combining efficient protein cleavage and high performance liquid chromatography separation with the sensitivity of mass spectrometry (MS), this technique allows the observation of the full protein map and the posttranslational modifications of the protein in a single experiment. The rhodopsin protein from a single porcine eye was sequenced completely, with the exception of two single-amino acid fragments and one two-amino acid fragment, and the gene sequence reported previously was confirmed. The posttranslational modifications, similar to the ones reported previously for bovine and rat rhodopsin, were also identified. Although porcine rhodopsin has a high degree of homology to bovine and rat rhodopsins and most of their posttranslational modifications are identical, the glycosylation and phosphorylation patterns observed were different. These results show that rhodopsin from a single porcine eye can be characterized completely by MS. This technology opens the possibility of rhodopsin structural and functional studies aided by powerful mass spectrometric analysis, using the fellow eye as an internal control.     

Toward high sequence coverage of proteins in human breast cancer cells using on-line monolith-based HPLC-ESI-TOF MS compared to CE MS

A method is developed toward high sequence coverage of proteins isolated from human breast cancer MCF10 cell lines using a 2-D liquid separations. Monolithic-capillary columns prepared by copolymerizing styrene with divinylbenzene are used to achieve high-resolution separation of peptides from protein digests. This separation is performed with minimal sample preparation directly from the 2-D liquid fractionation of the cell lysate. The monolithic column separation is directly interfaced to ESI-TOF MS to obtain a peptide map. The protein digests were also analyzed by MALDI-TOF MS and an accurate M(r) of the intact protein was obtained using an HPLC-ESI-TOF MS. The result is that these techniques provide complementary information where nearly complete sequence coverage of the protein is obtained and can be compared to the experimental M(r) value. The high sequence coverage provides information on isoforms and other post-translational modifications that would not be available from methods that result in low sequence coverage. The results from the use of monolithic columns are compared to that obtained by CE-MS. The monolithic column separations provide a rugged and highly reproducible method for separating protein digests prior to MS analysis and is suited to confidently identify biomarkers associated with cancer progression.     

一种改进的凝胶分离后蛋白质溶液酶解方法

报道了改进的凝胶电泳分离后蛋白质溶液酶解方法。将凝胶分离后的蛋白转移到PVDF膜上,直接用碳酸氢铵中和从膜上的蛋白洗脱溶液至弱碱性,并在此溶液中直接进行蛋白酶解。方法避免了胶内杂质对质谱鉴定蛋白质的干扰。用标准蛋白进行了验证,并在此基础上鉴定了中国仓鼠卵巢细胞（CHO）细胞系中的16个蛋白点。与已报道的方法比较,本方法蛋白回收率提高到90％左右;与传统的胶内酶解方法比较,本方法具操作简单,实验流程较短;涉及的试剂较胶内酶解少,因此带来的杂质影响小;在蛋白转印以后还可与许多其它实验方法相结合,比如免疫印迹等优点。     

MALDI‐TOF mass spectrometry of hordeins: rapid approach for identification of malting barley varieties

A procedure for identification of malting barley varieties using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) of ethanol‐soluble barley proteins (hordeins) is described. The hordeins were first extracted from milled barley grains by several extraction protocols (using different extraction agents and conditions). Hordein extracts were then analyzed directly via MALDI‐TOF MS without any preliminary purification or separation step, and the protein profiles of analyzed hordein extracts were compared in order to find out the most suitable extraction procedure for mass spectrometric analysis. The optimized procedure was successfully applied to identification of 13 malting barley varieties. Our results revealed that the proposed mass spectrometry‐based approach provides characteristic mass patterns of extracted hordeins, which can be advantageously used for barley variety identification. Copyright © 2009 John Wiley & Sons, Ltd.     

Proteomic profiling of a snake venom using high mass detection MALDI-TOF mass spectrometry

Proteomic profiling involves identification and quantification of protein components in complex biological systems. Most of the mass profiling studies performed with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) have been restricted to peptides and small proteins (<20 kDa) because the sensitivity of the standard ion detectors decreases with increasing ion mass. Here we perform a protein profiling study of the snake venom Sistrurus miliarius barbouri, comparing 2D gel electrophoresis and reversed-phase high-performance liquid chromatography (HPLC) with a high mass cryodetector MALDI-TOF instrument (Macromizer), whose detector displays an uniform sensitivity with mass. Our results show that such MS approach can render superior analysis of protein complexity compared with that obtained with the electrophoretic and chromatographic approaches. The summation of ion impacts allows relative quantification of different proteins, and the number of ion counts correlates with the peak areas in the reversed-phase HPLC. Furthermore, the sensitivity reached with the high mass cryodetection MS technology clearly exceeds the detection limit of standard high-sensitivity staining methods.     

Identification of 2D-gel proteins: a comparison of MALDI/TOF peptide mass mapping to mu LC-ESI tandem mass spectrometry

A comparative analysis of protein identification for a total of 162 protein spots separated by two-dimensional gel electrophoresis from two fully sequenced archaea, Methanococcus jannaschii and Pyrococcus furiosus, using MALDI-TOF peptide mass mapping (PMM) and mu LC-MS/MS is presented. 100% of the gel spots analyzed were successfully matched to the predicted proteins in the two corresponding open reading frame databases by mu LC-MS/MS while 97% of them were identified by MALDI-TOF PMM. The high success rate from the PMM resulted from sample desalting/concentrating with ZipTip(C18) and optimization of several PMM search parameters including a 25 ppm average mass tolerance and the application of two different protein molecular weight search windows. By using this strategy, low-molecular weight (<23 kDa) proteins could be identified unambiguously with less than 5 peptide matches. Nine percent of spots were identified as containing multiple proteins. By using mu LC-MS/MS, 50% of the spots analyzed were identified as containing multiple proteins. mu LC-MS/MS demonstrated better protein sequence coverage than MALDI-TOF PMM over the entire mass range of proteins identified. MALDI-TOF and PMM produced unique peptide molecular weight matches that were not identified by mu LC-MS/MS. By incorporating amino acid sequence modifications into database searches, combined sequence coverage obtained from these two complimentary ionization methods exceeded 50% for approximately 70% of the 162 spots analyzed. This improved sequence coverage in combination with enzymatic digestions of different specificity is proposed as a method for analysis of post-translational modification from 2D-gel separated proteins.     

High-resolution separations of protein isoforms with liquid chromatography time-of-flight mass spectrometry using polymer monolithic capillary columns

The separation of intact proteins, including protein isoforms arising from various amino-acid modifications, employing a poly(styrene-co-divinylbenzene) monolithic capillary column in high-performance liquid chromatography coupled on-line to a time-of-flight mass spectrometer (MS) is described. Using a 250 mm × 0.2 mm monolithic capillary column high-sensitivity separations yielding peak capacities of >600 were achieved with a 2h linear gradient and formic acid added in the mobile phase as ion-pairing agent. The combination of high-resolution chromatography with high-accuracy MS allowed to distinguish protein isoforms that differ only in their oxidation and biotinylation state allowing the separation between structural isoforms. Finally, the potential to separate proteins isoforms due to glycosylation is discussed.     

Ultra-fast tandem mass spectrometry scanning combined with monolithic column liquid chromatography increases throughput in proteomic analysis

Liquid chromatography combined with electrospray ionization mass spectrometry (LC/ESI-MS) has been used successfully for the characterization of biomolecules in proteomics in the last few years. This methodology relied largely on the use of reversed-phase chromatography, in particular C18-based resins, which are suitable for separation of peptides. Here we show that polymeric [polystyrene divinylbenzene] monolithic columns can be used to separate peptide mixtures faster and at a higher resolution. For 500 fmol bovine serum albumin, up to 68% sequence coverage and Mascot Mowse scores of >2000 were obtained using a 9 min gradient on a monolithic column coupled to an ion trap mass spectrometer with ultra-fast MS/MS scan rates. In order to achieve similar results using C18 columns, it was necessary to extend gradient times to 30 min. In addition, we demonstrate the utility of this approach for the analysis of whole Escherichia coli cell lysates by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) in combination with LC/MS/MS using 4 min gradients on monolithic columns. Our results indicate higher throughput capabilities of monolithic columns (3-fold gain in time or more) for conventional proteomics applications, such as protein identification and high sequence coverage usually required for detection of post-translational modifications (PTMs). Further optimization of sensitivity and quality of sequence information is discussed, in particular when combined with mass spectrometers that have very fast MS-MS/MS switching and scanning capabilities.     

Identification of phosphorylation and acetylation sites in αA-crystallin of the eye lens (<Emphasis Type="Italic">mus musculus</Emphasis>) after two-dimensional gel electrophoresis

Posttranslational modifications are of great interest because of their relevance in biological systems as proteins are commonly activated or deactivated by phosphorylation, glycation and acetylation [1, 2]. During eye lens aging the number of the A-crystallin isoproteins increases. This could be observed by the use of 2D-PAGE (two-dimensional gel electrophoresis). The number of A-crystallin spots in the gel increased during eye lens aging. For further analysis the spots of 2D-PAGE were cut out and the identification of the proteins was done using nanoLC-ESI-MS/MS (liquid chromatography electrospray ionization tandem mass spectrometry). The created MS/MS-data were analyzed using the Sequest algorithm. Searches with different parameters were done to preferably get the complete sequence coverage and to identify posttranslational modifications of the A-crystallin. The acetylated N-terminus of this protein could be detected. Furthermore, phosphorylation of serine 122 and 148 was identified in two different spots.     

Determination of the glycation sites of Bacillus anthracis neoglycoconjugate vaccine by MALDI-TOF/TOF-CID-MS/MS and LC-ESI-QqTOF-tandem mass spectrometry

We present herein an efficient mass spectrometric method for the localization of the glycation sites of a model neoglycoconjugate vaccine formed by a construct of the tetrasaccharide side chain of the Bacillus anthracis exosporium and the protein carrier bovine serum albumin. The glycoconjugate was digested with both trypsin and GluC V8 endoproteinases, and the digests were then analyzed by MALDI-TOF/TOF-CID-MS/MS and nano-LC-ESI-QqTOF-CID-MS/MS. The sequences of the unknown peptides analyzed by MALDI-TOF/TOF-CID-MS/MS, following digestion with the GluC V8 endoproteinase, allowed us to recognize three glycopeptides whose glycation occupancies were, respectively, on Lys 235, Lys 420, and Lys 498. Similarly, the same analysis was performed on the tryptic digests, which permitted us to recognize two glycation sites on Lys 100 and Lys 374. In addition, we have also used LC-ESI-QqTOF-CID-MS/MS analysis for the identification of the tryptic digests. However, this analysis identified a higher number of glycopeptides than would be expected from a glycoconjugate composed of a carbohydrate-protein ratio of 5.4:1, which would have resulted in glycation occupancies of 18 specific sites. This discrepancy was due to the large number of glycoforms formed during the synthetic carbohydrate-spacer-carrier protein conjugation. Likewise, the LC-ESI-QqTOF-MS/MS analysis of the GluC V8 digest also identified 17 different glycation sites on the synthetic glycoconjugate.     

Posttranslational modifications of collagen studied by off-line coupling of HPLC and CE

A typical example of non-enzymatic change of collagen is glycation (the Maillard reaction, formation of advanced glycation end products) resulting from the reaction of sugars with the epsilon-amino group of lysine. Posttranslational non-enzymatic modifications of collagen by sugars were studied. Collagenous tissues were incubated as a test protein separately with both glucose and ribose. The collagen mixture was digested by bacterial collagenase and separated by reversed-phase HPLC (in a Jupiter Proteo 90 A column). The eluate from this HPLC separation was collected as seven fractions and consecutively analysed by CE in a bare fused silica capillary (57/50 cm x 75 mm id) using 100 mM sodium 1-heptanesulfonate in 100 mM phosphate buffer, pH 2.5 (NaH2PO4 adjusted to pH by phosphoric acid). The chromatographic and electromigration behaviour of individual peptides varied considerably. This off-line HPLC-CE coupling made it possible to discover minor changes in the structure of collagen caused by posttranslational modifications. A new HPLC-CE technique for peptide analysis was developed, and applied to the identification of posttranslational modifications in slowly metabolised test proteins.     

Mass spectrometric detection of CP4 EPSPS in genetically modified soya and maize

The potential of protein fractionation hyphenated to mass spectrometry (MS) to detect and characterize the transgenic protein present in Roundup Ready soya and maize has been investigated. Genetically modified (GM) soya and maize contain the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from Agrobacterium tumefaciens CP4, which confers resistance to the herbicide glyphosate. The GM soya and maize proteomes were fractionated by gel filtration, anion-exchange chromatography and sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) prior to MS. This facilitated detection of a tryptic peptide map of CP4 EPSPS by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and nanoelectrospray ionization quadrupole time-of-flight (nanoESI-QTOF) MS. Subsequently, sequence information from the CP4 EPSPS tryptic peptides was obtained by nanoESI-QTOF MS/MS. The identification was accomplished in 0.9% GM soya seeds, which is the current EU threshold for food-labeling requirements.     

磺化修饰结合离子交换色谱和生物质谱富集鉴定含组氨酸肽段

通过在肽段的N端引入磺酸基,从而使含组氨酸的肽段与其他肽段在pH<3.0的条件下产生电荷差异,建立了一种基于强阳离子交换色谱(SCX)结合生物质谱富集鉴定含组氨酸肽段的方法,并以含有组氨酸的标准蛋白质为模型,进行了方法学考察。结果表明,经N端磺酸化后,含组氨酸的肽段能有效地被阳离子交换色谱富集,且在肽的N端引入磺酸基促进了肽的裂解,使之产生简单而信息丰富的二级质谱图,从而得到完美的质谱鉴定结果。这说明磺化修饰结合强阳离子交换色谱用于含组氨酸肽段的富集鉴定是可行的,且具有在蛋白质组研究中应用的潜力。