Alternative mobile phases for enhanced chromatographic selectivity and increased sensitivity in peptide separations.

The standard method for separating peptide mixtures is reversed-phase high-performance liquid chromatography with gradients of increasing concentrations of acetonitrile in the presence of trifluoroacetic acid. With modern instruments and columns, complex peptide mixtures can be separated, and low picomole amounts can be collected in tens of microliters. Difficult separations are addressed by modifying the gradient slope or organic eluant composition. Further improvements in resolution are often needed, requiring fundamental changes in mobile phase composition or selection of complementary chromatographic separation mechanisms. For the present study, tryptic digests of cytochrome c from various species were separated in the presence of dilute hydrochloric acid by reversed phase on a Waters Delta-PakTM C18 high-performance insert column and by strong cation exchange on a Waters Protein-PakTM SP 8HR. Different and enhanced reversed-phase selectivity was obtained by replacing trifluoroacetic acid with dilute hydrochloric acid at the same pH. The increased optical clarity of hydrochloric acid-based mobile phases in the low ultraviolet wavelengths yielded increased sensitivity. Very different selectivity was observed with the cation-exchange chromatography. These data expand the options for peptide mapping by providing additional selectivity combined with increased mass sensitivity and spectral information in the low ultraviolet.     

Isolation and quantification of dinucleoside polyphosphates by using monolithic reversed phase chromatography columns

In former studies, dinucleoside polyphosphates were quantified using ion-pair reversed-phase perfusion chromatography columns, which allows a detection limit in the micromolar range. The aim of this study was both to describe a chromatographic assay with an increased efficiency of the dinucleoside separation, which enables the reduction of analytical run times, and to establish a chromatographic assay using conditions, which allow MALDI-mass spectrometric analysis of the resulting fractions. We compared the performance of conventional silica reversed phase chromatography columns, a perfusion chromatography column and a monolithic reversed-phase C18 chromatography column. The effects of different ion-pair reagents, flow-rates and gradients on the separation of synthetic diadenosine polyphosphates as well as of diadenosine polyphosphates isolated from human platelets were analysed. Sensitivity and resolution of the monolithic reversed-phase chromatography column were both higher than that of the perfusion chromatography and the conventional reversed phase chromatography columns. Using a monolithic reversed-phase C18 chromatography column, diadenosine polyphosphates were separable baseline not only in the presence of tetrabutylammonium hydrogensulfate (TBA) but also in the presence of triethylammonium acetate (TEAA) as ion-pair reagent. The later reagent is useful because, in contrast to TBA, it is compatible with MALDI mass-spectrometric methods. This makes TEAA particularly suitable for identification of unknown nucleoside polyphosphates. Furthermore, because of the lower backpressure of monolithic reversed-phase chromatography columns, we were able to significantly increase the flow rate, decreasing the amount of time for the analysis close to 50%, especially using TBA as ion-pair reagent. In summary, monolithic reversed phase C18 columns markedly increase the sensitivity and resolution of dinucleoside polyphosphate analysis in a time-efficient manner compared to reversed-phase perfusion chromatography columns or conventional reversed-phase columns. Therefore, further dinucleoside polyphosphate analytic assays should be based on monolithic silica C18 columns instead of perfusion chromatography or conventional silica reversed phase chromatography columns. In conclusion, the use of monolithic silica C18 columns will lead to isolation and quantification of up to now unknown dinucleoside polyphosphates. These chromatography columns may facilitate further research on the biological roles of dinucleoside polyphosphates.     

Systematic Separation of Human Urinary Peptides

Urinary peptides were roughly fractionated by combined columns of cation and anion exchange resins, and the peptides eluted from each column were further fractionated by a combination of various ion exchange resins and DEAE-cellulose column chromatography, paper chromatography and other methods. From the fractions adsorbed on cation exchange resin, 13 homogeneous peptides could be isolated, and from the ones adsorbed on anion exchange resin, 8 glycopeptides could be found. Their amino acid compositions were analyzed.

Although some fractions remain univestigated, an outline of the whole aspect of main urinary peptides has been clarified by this study.     

Continuous desalting of refolded protein solution improves capturing in ion exchange chromatography: A seamless process

Truly continuous biomanufacturing processes enable an uninterrupted feed stream throughout the whole production without the need for holding tanks. We have utilized microporous anion and cation exchangers into which only salts, but not proteins, can penetrate into the pores for desalting of protein solutions, while diafiltration or dilution is usually employed for feed adjustments. Anion exchange and cation exchange chromatography columns were connected in series to remove both anions and cations. To increase operation performance, a continuous process was developed comprised of four columns. Continuous mode was achieved by staggered cycle operation, where one set of columns, consisting of one anion exchange and one cation exchange column, was loaded during the regeneration of the second set. Refolding, desalting and subsequent ion exchange capturing with a scFv as the model protein was demonstrated. The refolding solution was successfully desalted resulting in a consistent conductivity below 0.5 mS/cm from initial values of 10 to 11 mS/cm. With continuous operation process time could be reduced by 39% while productivity was increased to 163% compared to batch operation. Desalting of the protein solution resulted in up to 7‐fold higher binding capacities in the subsequent ion exchange capture step with conventional protein binding resins.     

Multiplexed two-dimensional liquid chromatography for MALDI and nanoelectrospray ionization mass spectrometry in proteomics

We developed a multiplexed two-dimensional separation system based on reversed phase (RP)--strong cation exchange (SCX) chromatography as a front-end device for matrix-assisted laser desorption ionization (MALDI) or nanoelectrospray ionization (nanoESI) mass spectrometry. Tryptic peptide mixtures were fractionated on a reversed-phase HPLC column, and each fraction was loaded onto multiplexed SCX microcolumns. Because this second chromatography was carried out in parallel, the analysis time is independent of the fraction number in the first RP-HPLC separation. The resultant samples were desalted/concentrated and eluted onto a MALDI plate with matrix-containing elution solutions in parallel, or eluted with optimized solutions for nanoESI and loaded onto nanoESI sprayers by an automated instrument. The soluble portion of HCT116 lysate was digested and fractionated using a 48-plexed chromatography system. Approximately 1000 unique peaks were detected in MALDI-MS with 3000 MS/MS spectra, while 724 peptides with ultrahigh peptide mass accuracy (sub-ppm error) were identified in nanoESI-FTICR mass spectrometry with five integrated selected ion monitoring scans. Since MS measurement with this off-line LC-LC approach is not restricted by continuous LC elution, it is expected to be useful especially in cases where repeated analysis with different scan modes or long-term data acquisition is required.     

Purification and characterization of a novel antimicrobial peptide from Brevibacillus laterosporus strain A60

A novel antimicrobial peptide, with molecular mass of 1602.0469Da, produced by Brevibacillus laterosporus strain A60 was isolated and purified from the soil of mango plants. The purification procedure consisted of ammonium sulfate precipitation, cation exchange chromatography on an HiTrap SP HP column, thin layer chromatography and High Performance Liquid Chromatography (HPLC) on C18 reversed-phase column. After the four isolation procedures, one peptide with antimicrobial activity was obtained and named BL-A60. The determination of the complete amino acid sequences of this peptide showed that it contains eleven amino acid residues, L-Y-K-L-V-K-V-V-L-N-M, and a choline connected to the N-terminal and a tenuazonic acid modified of the C-terminal. This peptide shows relatively low identification to other antimicrobial peptides from bacteria. Purified BL-A60 showed high pH and thermal stability and a strong inhibition of different stages of the life cycle of Phytophthora capsici, including mycelial growth, sporangia formation and cystospore germination, with EC(50) values of 7.89, 0.60 and 21.96 μg ml(-1), respectively.     

Correlations between in vitro potency of polyethylene glycol-protein conjugates and their chromatographic behavior

Pegylation is the most widely used and accepted methodology for half-life extension of biopharmaceutical drugs that also improves physicochemical and biological characteristics of proteins considerably. Most of the positive pharmacological effects of pegylated proteins are believed to be related to an increased hydrodynamic volume and molecular size. To explore the size impact of polyethylene glycol (PEG) on in vitro potency, a series of well-defined conjugates of interferon α-2b (IFN) were prepared with PEGs of different lengths and shapes specifically attached to the N-terminal amino group of the protein. Specificity of the attachment was confirmed by peptide mapping and mass spectroscopy. When potency values determined by reporter gene assay were correlated with methods for molecular weight and size characterization, such as size exclusion chromatography and dynamic light scattering, rough parallels were found. Unexpectedly, the retention times on cation exchange chromatography showed much higher correlation with experimentally determined in vitro potency. It appears that in a series of N-terminally pegylated IFNs, their in vitro potency could be predicted from the retention times on the cation exchange chromatography columns, probably because both methods reflect not only the influence of molecular size but also the impact of protein masking exerted by attached PEG moiety.     

Reversible interference of Fe³(+) with monoclonal antibody analysis in cation exchange columns

The effect of Fe3(+) treatment on weak cation exchange column chromatography was demonstrated for monoclonal antibody (MAb) analysis. Fe3(+)-exposed columns showed lowered relative peak areas of the parent MAb peak as well as both acidic and basic variant peaks that could lead to erroneous conclusions. Accurate measurement of relative amounts of variants to the parent MAb is essential for demonstrating the safety and efficacy of therapeutic molecules such as MAbs. Complete reversal of the compromised MAb analysis performance was observed after washing the column with chelating agents, confirming that metal contamination was responsible for the compromised column performance.     

Efficient purification of small unsaturated oligoglucuronides by reversed-phase chromatography

Ion-exchange chromatography has been applied to purification of unsaturated oligoglucuronans. After an isocratic elution on a strong anion-exchange column, the collected fractions were desalted by low pressure size exclusion chromatography. However, this efficient separation was limited by the time required to desalt. So, we developed a reversed-phase chromatography method using back ionization of oligomers. Two C18 columns were tested with trifluoroacetic acid (TFA 0.7%) as eluent. Different selectivities and column stabilities were observed in this acidic condition. The scale up for semi-preparative applications enabled us to recover pure unsaturated oligoglucuronans without desalting step.     

Characterization of immunoreactive motilin from the rat small intestine.

Immunocytochemistry, radioimmunoassay, chromatography, and biological assay using a rabbit isolated duodenal muscle strip preparation were used in attempting to characterize motilin from the rat small intestine. Several different antisera and monoclonal antibodies directed against natural porcine motilin were used. A variety of fixation techniques using Bouin's, paraformaldehyde, and benzoquinone with different staining methods including, fluorescein-conjugated second antibody, peroxidase-antiperoxidase or peroxidase-conjugated second antibody techniques were used. All methods failed to detect immunoreactive motilin cells in the rat small intestine. The same antisera were used in radioimmunoassays for motilin to evaluate extracts of rat intestinal tissue. Two of these detected immunoreactive motilin in gut extracts, and these antisera showed a different distribution for the peptide. Samples containing immunoreactive motilin obtained from cation exchange chromatography on SP-Sephadex-G25 were concentrated and assayed for biological activity in a rabbit duodenal muscle strip preparation. Desensitization of duodenal tissue to porcine motilin could be demonstrated by pretreatment with this peptide. The biological activity of partially purified rat intestinal immunoreactive motilin was not prevented by pretreatment of the tissue with motilin. Further purification of this preparation on Bio-Gel P-10 yielded an immunoreactive motilin peak that co-eluted with natural porcine motilin. Rat intestinal immunoreactive motilin did not co-elute with natural porcine motilin following high pressure liquid chromatography on a Waters microBondapak C18 reversed-phase column using a linear gradient of water-acetonitrile (10-45%) over 30 min. Although of similar molecular size, rat motilin is probably structurally dissimilar to other mammalian motilins.     

Fractionation of complex protein mixture by virtual three-dimensional liquid chromatography based on combined pH and salt steps

The complexity and diversity of biological samples in proteomics require intensive fractionation ahead of mass spectrometry identification. This work developed a chromatographic method called virtual three-dimensional chromatography to fractionate complex protein mixtures. By alternate elution with different pHs and salt concentrations, we implemented pH and salt steps by turns on a single strong cation exchange column to fully exploit its chromatographic ability. Given standard proteins that were not resolved solely by pH or salt gradient elution could be successfully separated using this combined mode. With a reversed phase column tandem connected behind, we further fractionated as well as desalted proteins as the third dimension. This present strategy could readily be adapted with respect to special complexity of biological samples. Crude plasma without depleting high abundance proteins were fractionated by this three-dimensional mode and then analyzed by reversed phase liquid chromatography coupled with LTQ mass spectrometry. In total, 1933 protein groups with wide dynamic ranges were identified from a single experiment. Some characteristics that correlated to the behavior of proteins on strong cation exchange columns are also discussed.     

Reversed-phase chromatography with multiple fraction concatenation strategy for proteome profiling of human MCF10A cells

In this study, we evaluated a concatenated low pH (pH 3) and high pH (pH 10) reversed-phase liquid chromatography strategy as a first dimension for two-dimensional liquid chromatography tandem mass spectrometry ("shotgun") proteomic analysis of trypsin-digested human MCF10A cell sample. Compared with the more traditional strong cation exchange method, the use of concatenated high pH reversed-phase liquid chromatography as a first-dimension fractionation strategy resulted in 1.8- and 1.6-fold increases in the number of peptide and protein identifications (with two or more unique peptides), respectively. In addition to broader identifications, advantages of the concatenated high pH fractionation approach include improved protein sequence coverage, simplified sample processing, and reduced sample losses. The results demonstrate that the concatenated high pH reversed-phased strategy is an attractive alternative to strong cation exchange for two-dimensional shotgun proteomic analysis.     

Comparison of protein and peptide prefractionation methods for the shotgun proteomic analysis of Synechocystis sp. PCC 6803

Proteome analysis by gel-free "shotgun" proteomics relies on the simplification of a peptide mixture before it is analyzed in a mass spectrometer. While separation on a reverse-phase (RP) liquid chromatographic column is widely employed, a variety of other methods have been used to fractionate both proteins and peptides before this step. We compared six different protein and peptide fractionation workflows, using Synechocystis sp. PCC 6803, a useful model cyanobacterium for potential exploitation to improve its production of hydrogen and other secondary metabolites. Pre-digestion protein separation was performed by strip-based isoelectric focusing, one-dimensional polyacrylamide gel electrophoresis, or weak anion exchange chromatography, while pre-RP peptide separation was accomplished by isoelectric focusing (IEF) or strong cation exchange chromatography. Peptides were identified using electrospray ionization quadrupole time of flight-tandem mass spectrometry. Mass spectrometry (MS) and tandem mass spectra were analyzed using ProID software employing both a single organism database and the entire NCBI non-redundant database, and a total of 776 proteins were identified using a stringent set of selection criteria. Method comparisons were made on the basis of the results obtained (number and types of proteins identified), as well as ease of use and other practical aspects. IEF-IEF protein and peptide fractionation prior to RP gave the best overall performance.     

A Review of: “Biological Recognition and Assembly,D. S. Eisenberg,J. A. Lake and C. F. Fox,Eds. Alan R. Liss,New York, 1980; hardbound, 355 pages, $52.00”

Two MMP-7-ase isoenzymes were purified 100-fold from rat muscle extract to apparent homogeneity, with an overall yield of 10%, using homogenization, ultracentrifugation, high-performance aqueous size-exclusion and high-performance anion exchange chromatography methods. When using a TSK G-2000SW column, the separation resulted in a 6-fold purification and 30% recovery of isoenzymes B and C. This concentrated enzyme extract was then passed through a TSK-DEAE-2SW column, using salt gradient at pH 7.5, with an additional 25-fold purification and 90% recovery of the isoenzymes. Two symmetrical enzyme peaks, representing isoenzymes B and C, were detected when performing purity tests of the active enzymes on the anion exchanger and reversed-phase HFLC columns. The procedures involved are extraction, ultracentri-fugation, chromatographies and enzyme assays and require less than five hours.     

Method for isolation of MMP-7-ase isoenzymes from rat muscle tissue extract

Two MMP-7-ase isoenzymes were purified 100-fold from rat muscle extract to apparent homogeneity, with an overall yield of 10%, using homogenization, ultracentrifugation, high-performance aqueous size-exclusion and high-performance anion exchange chromatography methods. When using a TSK G-2000SW column, the separation resulted in a 6-fold purification and 30% recovery of isoenzymes B and C. This concentrated enzyme extract was then passed through a TSK-DEAE-2SW column, using salt gradient at pH 7.5, with an additional 25-fold purification and 90% recovery of the isoenzymes. Two symmetrical enzyme peaks, representing isoenzymes B and C, were detected when performing purity tests of the active enzymes on the anion exchanger and reversed-phase HPLC columns. The procedures involved are extraction, ultracentrifugation, chromatographies and enzyme assays and require less than five hours.     

Protein phosphorylation and expression profiling by Yin-yang multidimensional liquid chromatography (Yin-yang MDLC) mass spectrometry

A system which consisted of multidimensional liquid chromatography (Yin-yang MDLC) coupled with mass spectrometry was used for the identification of peptides and phosphopeptides. The multidimensional liquid chromatography combines the strong-cation exchange (SCX), strong-anion exchange (SAX), and reverse-phase methods for the separation. Protein digests were first loaded on an SCX column. The flow-through peptides from SCX were collected and further loaded on an SAX column. Both columns were eluted by offline pH steps, and the collected fractions were identified by reverse-phase liquid chromatography tandem mass spectrometry. Comprehensive peptide identification was achieved by the Yin-yang MDLC-MS/MS for a 1 mg mouse liver. In total, 14 105 unique peptides were identified with high confidence, including 13 256 unmodified peptides and 849 phosphopeptides with 809 phosphorylated sites. The SCX and SAX in the Yin-Yang system displayed complementary features of binding and separation for peptides. When coupled with reverse-phase liquid chromatography mass spectrometry, the SAX-based method can detect more extremely acidic (pI < 4.0) and phosphorylated peptides, while the SCX-based method detects more relatively basic peptides (pI > 4.0). In total, 134 groups of phosphorylated peptide isoforms were obtained, with common peptide sequences but different phosphorylated states. This unbiased profiling of protein expression and phosphorylation provides a powerful approach to probe protein dynamics, without using any prefractionation and chemical derivation.     

Reversible interference of Fe with monoclonal antibody analysis in cation exchange columns

The effect of Fe³⁺ treatment on weak cation exchange column chromatography was demonstrated for monoclonal antibody (MAb) analysis. Fe³⁺-exposed columns showed lowered relative peak areas of the parent MAb peak as well as both acidic and basic variant peaks that could lead to erroneous conclusions. Accurate measurement of relative amounts of variants to the parent MAb is essential for demonstrating the safety and efficacy of therapeutic molecules such as MAbs. Complete reversal of the compromised MAb analysis performance was observed after washing the column with chelating agents, confirming that metal contamination was responsible for the compromised column performance.     

Quantitative determination of 5-hydroxytryptophan in dissected brain regions by on-line trace enrichment HPLC with electrochemical detection

A sensitive, specific and rapid quantitative HPLC assay for 5-hydroxytryptophan (5-HTP) in samples of brain regions of widely differing size is described. The method utilizes off-line prepurification of tissue supernatants on gravity-fed strong cation exchange columns, on-line enrichment of the entire cation exchange column eluate on short reverse phase enrichment precolumns, further separation by reverse phase chromatography on an analytical column and electrochemical detection. On-line trace enrichment permits the efficient incorporation of off-line column chromatography to maximize assay specificity without compromising assay sensitivity. A reliable, working limit of detection of 200 pg 5-HTP/sample permits the estimation of in vivo tryptophan hydroxylase activity by determining the rate of 5-HTP accumulation following L-aromatic amino acid decarboxylase inhibition in small discrete brain regions or larger tissue samples only poorly innervated by 5-HT terminals.     

Capillary array reversed-phase liquid chromatography-based multidimensional separation system coupled with MALDI-TOF-TOF-MS detection for high-throughput proteome analysis

A high-throughput on-line capillary array-based two-dimensional liquid chromatography (2D-LC) system coupled with MALDI-TOF-TOF-MS proteomics analyzer for comprehensive proteomic analyses has been developed, in which one capillary strong-cation exchange (SCX) chromatographic column was used as the first separation dimension and 18 parallel capillary reversed-phase liquid chromatographic (RPLC) columns were integrated as the second separation dimension. Peptides bound to the SCX phase were "stepped" off using multiple salt pulses followed by sequentially loading of each subset of peptides onto the corresponding precolumns. After salt fractionation, by directing identically split solvent-gradient flows into 18 channels, peptide fractions were concurrently back-flushed from the precolumns and separated simultaneously with 18 capillary RP columns. LC effluents were directly deposited onto the MALDI target plates through an array of capillary tips at a 15-s interval, and then alpha-cyano-4-hydroxycinnamic acid (CHCA) matrix solution was added to each sample spot for subsequent MALDI experiments. This new system allows an 18-fold increase in throughput compared with serial-based 2D-LC system. The high efficiency of the overall system was demonstrated by the analysis of a tryptic digest of proteins extracted from normal human liver tissue. A total of 462 proteins was identified, which proved the system's promising potential for high-throughput analysis and application in proteomics.     

Two-dimensional reversed-phase x ion-pair reversed-phase HPLC: an alternative approach to high-resolution peptide separation for shotgun proteome analysis

A two-dimensional separation scheme for shotgun proteome analysis employing high-pH reversed-phase HPLC in the first and low-pH ion-pair reversed-phase HPLC in the second dimension (RP x IP-RP-HPLC) has been developed and evaluated. Compared to the classical strong cation exchange x ion-pair reversed-phase (SCX x IP-RP-HPLC) approach, the RP x IP-RP-HPLC system was characterized by a lower degree of orthogonality, which was, however, more than counterbalanced by higher separation efficiency, more homogeneous distribution of peptide elution, and easier experimental handling. Peptide fragment fingerprinting by electrospray-ionization tandem mass spectrometry (ESI-MS/MS) was employed for peptide detection and identification. About 13% more peptides and 7% more proteins could be identified with the alternative approach in 30% less analysis time, enabling the analysis of the proteome of Corynebacterium glutamicum with a coverage of 24.9% (745 proteins). Combining the identification results both of the SCX- x IP-RP-HPLC-ESI-MS/MS and RP- x IP-RP-HPLC-ESI-MS/MS methods, a total of 871 proteins were identified in a cytosolic protein preparation, which represented 29.1% of all proteins annotated in the genome of C. glutamicum.