Comprehensive analysis of host cell impurities in monoclonal antibodies with improved sensitivity by capillary zone electrophoresis mass spectrometry

Four methods were compared for analysis of host‐cell protein (HCP) impurities in a recombinant mAb. First, CZE‐MS/MS was used to analyze the digest of an HCP sample following extraction of the mAb with proteins A and L affinity columns; 220 protein groups and 976 peptides were identified from the depleted HCP digest. Second, a nanoACQUITY UltraPerformance LCH system was also used to analyze the depleted HCP digest; 34 protein groups and 53 peptides from 50 ng of the depleted HCP digest and 290 protein groups and 1011 peptides were identified from 1 μg of the depleted HCP digest. Third, 185 protein groups and 709 peptides were identified by CZE‐MS/MS from the HCP digest without depletion. Fourth, a strong cation exchange SPE was coupled to CZE‐ESI‐MS/MS using online pH gradient elution for analysis of the HCP digest without depletion. A series of five pH bumps were applied to elute peptides from the strong cation exchange monolith followed by analysis using CZE coupled to a Q Exactive HF mass spectrometer; 230 protein groups and 796 peptides were identified from the HCP digest without depletion.     

Quantitative Transfer of In-Gel Digest Products to Liquid Chromatography-Electrospray Mass Spectrometry Using On-Line Coupled Extraction

A system is described for the on-line extraction of a digested protein in a gel spot. The extract obtained was on-line transferred to a precolumn, used to desalinate and preconcentrate the sample. The precolumn was switched in-line with an LC-ESI-MS system to separate the digest products prior to detection. By using this approach sample losses were minimized and the sample was quantitatively transferred. The benefits of the system were shown for the in-gel tryptic digest of several model proteins. Using this system, the sensitivity was 13-fold increased compared to the off-line extraction of in-gel digest products leading to a higher sequence coverage and thus a better identification potential. Also, the within day repeatability was a factor 2.5 improvement using the on-line procedure compared to the off-line extraction procedure.     

Strategies for locating disulfide bonds in a monoclonal antibody via mass spectrometry

The location of the disulfide bonds in a recombinant monoclonal antibody was confirmed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) and electrospray ionization (ESI) mass spectrometry (MS). A non-reduced Endoproteinase Lys-C (Endo Lys-C) digest of the antibody was analyzed directly by MALDI-TOFMS. The sample was then reduced on-plate by depositing dithiothreitol (DTT) on the sample spot and re-analyzed by MALDI-TOFMS. The disulfide bonds were assigned based on the disappearance of certain mass ions in the non-reduced digest and the appearance of product ions in the reduced digest. A rapid LC/ESI-MS protocol was also developed to determine the location of the disulfide bonds. The peptides generated from the Endo Lys-C digest of the antibody were partially separated on a high performance liquid chromatography (HPLC) column by utilizing a steep gradient and analyzed by ESI-MS. The masses of the partially resolved peptides were determined by deconvoluting the mass spectra.     

Mass mapping analysis as a tool for the identification of genetic variants of bovine beta-casein

Matrix-assisted laser desorption ionisation time-of-flight mass spectrometric analysis of the tryptic digest of beta-casein A2 and beta-casein B was performed before and after the separation of the peptides by LC. The overlapping of the chromatograms showed that all peaks were present in both samples, except for one only found in the tryptic digest of the A2 variant and two in the B variant. Experimental masses could be assigned to those peptides produced by tryptic digest of beta-casein variant. This peptide mapping strategy and current methodological improvements represent a promising tool for the identification of milk genetic variants with the difference of an amino acid substitution.     

东亚马氏钳蝎哺乳动物类神经毒素III的快原子轰击酶谱分析

应用快原子轰击酶谱分析检验了东亚马氏钳蝎哺乳动物类神经毒素III的氨基酸序列,对前人用Edman降解法测定的序列作了两处修正:(1)第60位氨基酸残基是脯氨酸而不是色氨酸;(2)C-端氨基酸是组氨酸而不是甘氨酸。分别用羧肽酶B降解和部分胰蛋白酶酶解后的质谱数据进一步证实了以上的修正。在分析中采用了化学修饰方法以减少表面抑制效应,从而得到完整的快原子轰击酶谱。     

The determination of glycopeptides by liquid chromatography/mass spectrometry with collision-induced dissociation

Glycopeptides derived from ribonuclease B and ovomucoid have been subjected to collision-induced dissociation (CID) in the second quadrupole of a triple quadrupole mass spectrometer. Doubly charged parent ions gave predictable fragmentation that yielded partial sequence information of the attached oligosaccharide as Hex and HexNAc units. Common oxonium ions are observed in the product ion mass spectra of the glycopeptides that correspond to HexNAc⁺ (m/z 204) and HexHexNAc⁺ (m/z 366). A strategy for locating the glycopeptides in the proteolytic digest mixtures of glycoproteins by ions spray liquid chromatography mass spectrometry (LC/MS) is described by utilizing CID in the declustering region of the atmospheric pressure ionization mass spectrometer to produce these characteristic oxonium ions. This LC/CID/MS approach is used to identify glycopeptides in proteolytic digest mixtures of ovomucoid, asialofetuin, and fetuin. LC/CID/MS in the selected ion monitoring mode may be used to identify putative glycopeptides from the proteolytic digest of fetuin.     

高温法和烘箱法测定水体中总氮的比较

分别采用烘箱法和高压蒸汽消解法对纯水和电解质溶液中的含氮有机化合物进行了消解,并用镉铜还原法测定了溶液中总氮的含量。实验表明,这两种消解方式无显著性差异,可以采用烘箱法代替压力蒸汽消毒器在电解质溶液中对含氮有机化合物进行消解,且烘箱法操作简单。     

Mass spectrometric analysis of enzymatic digestion of denatured collagen for identification of collagen type

Collagen type II and I from bovine were thermally denatured and digested with trypsin. The digest mixture was analyzed with liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). Peptides in the digest mixture were identified by mass spectrometry/mass spectrometry (MS/MS) sequencing. The results indicated that the digest mixtures of collagen type II and I contained lots of specific peptides and common peptides. Specific peptides could be used as index for identifying collagen type. Articular cartilage from bovine was pretreated and analyzed with the same method to determine the collagen types. The result indicated that the method developed was effective for identification of collagen types. The research provided a possible approach for collagen identification in particular tissues.     

Recent topics of the stereodivergent synthesis of natural products

A variety of natural products, a valuable source of drug lead compounds, coexist with their stereoisomers as congeners. For pursuing the structural elucidation and the structure–activity relationship study of natural products, it is needed to establish the streamlined synthetic route to supply natural products and their stereoisomers. Divergent pathway is one of the synthetic strategies to deliver more than one target compound. In this digest, selected examples of the stereodivergent approach toward the synthesis of natural products are described. Especially, this digest focuses on common synthetic intermediates and stereodiversification steps from the common intermediates to reach the target compounds.     

High‐resolution peptide separations using nano‐LC at ultra‐high pressure

We report on the optimization of nano‐LC gradient separations of proteomic samples that vary in complexity. The gradient performance limits were visualized by kinetic plots depicting the gradient time needed to achieve a certain peak capacity, while using the maximum system pressure of 80 MPa. The selection of the optimal particle size/column length combination and corresponding gradient steepness was based on scouting the performance of 75 μm id capillary columns packed with 2, 3, and 5 μm fully porous silica C18 particles. At optimal gradient conditions, peak capacities up to 500 can be obtained within a 120 min gradient using 2 μm particle‐packed capillary columns. Separations of proteomic samples including a cytochrome c tryptic digest, a bovine serum albumin tryptic digest, a six protein mix digest, and an Escherichia coli digest are demonstrated while operating at the kinetic‐performance limit, i.e. using 2‐μm packed columns, adjusting the column length and scaling the gradient steepness according to sample complexity. Finally, good run‐to‐run retention time stability (RSD values below 0.18%) was demonstrated applying ultra‐high pressure conditions.     

THE PRESENCE OF PHOTOREACTIVE β-CARBOLINE FLUOROPHORES IN WEATHERED WOOL KERATIN

Abstract Solvent-cleaned Merino wool, which was enzymatically digested with protease K, displayed a fluorescence maximum at 500 nm when excited at 430 nm. The yield of this emission was approximately 15 times greater for a fiber tips digest than for a digest of the mid-length region of the same fibers.
Separation of the components in the wool tip digest by silica gel thin-layer chromatography revealed the presence of several fluorescent species. The chromatographic mobility of these species was similar to the behavior observed in a preparation of authentic 1-methyltetrahydro-P-carboline, 1,3-dicarboxylic acid.
Mass spectroscopy of the fluorescent compounds in the wool tip digest displayed molecular ions, [M + H]⁺ with m/z = 273.0890 and 257.0560, and molecular weights that define the formulae C₁₄H₁₂N₂O₄ and C₁₁H₈O₄N₂, respectively, which correspond to β-carboline 1,3-dicarboxylic acids. Mass spectral evidence also indicates the presence of two other β-carbolines.     

Matrix-assisted laser desorption/ionization mass spectrometry screening for pseudouridine in mixtures of small RNAs by chemical derivatization, RNase digestion and signature products

We have developed a method to screen for pseudouridines in complex mixtures of small RNAs using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). First, the unfractionated crude mixture of tRNAs is digested to completion with an endoribonuclease, such as RNase T1, and the digestion products are examined using MALDI-MS. Individual RNAs are identified by their signature digestion products, which arise through the detection of unique mass values after nuclease digestion. Next, the endonuclease digest is derivatized using N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMCT), which selectively modifies all pseudouridine, thiouridine and 2-methylthio-6-isopentenyladenosine nucleosides. MALDI-MS determination of the CMCT-derivatized endonuclease digest reveals the presence of pseudouridine through a 252 Da mass increase over the underivatized digest. Proof-of-concept experiments were conducted using a mixture of Escherichia coli transfer RNAs and endoribonucleases T1 and A. More than 80% of the expected pseudouridines from this mixture were detected using this screening approach, even on an unfractionated sample of tRNAs. This approach should be particularly useful in the identification of putative pseudouridine synthases through detection of their target RNAs and can provide insight into specific small RNAs that may contain pseudouridine.     

Dynamic pH barrage junction focusing of amino acids,peptides, and digested monoclonal antibodies in capillary electrophoresis–mass spectrometry

Dynamic pH barrage junction focusing in CE enables effective signal enhancement, quantitative capture efficiencies, and straightforward optimization. The method is a technical variant of dynamic pH junction focusing. CE separation with dynamic pH barrage junction focusing is compatible with both optical and mass spectrometric detection. We developed a CE–MS/MS method using hydrophilic polyethyleneimine-coated capillaries and validated it for the qualitative analysis of amino acids, peptides, and tryptic peptides of digested monoclonal antibodies. The S/N of extracted ion electropherograms of zwitterionic analytes were enhanced by approximately two orders of magnitude with a tradeoff of a shortened separation window. Online focusing improved the MS signal intensity of a diluted antibody digest, enabling more precursor ions to be analyzed with subsequent tandem mass spectrometric identification. It also broadened the concentration range of protein digest samples for which adequate sequence coverage data can be obtained. With only 0.9 ng of digested infliximab sample loaded into the capillary, 76% and 100% sequence coverage was realized for antibody heavy and light chains, respectively, after online focusing. Full coverage was achieved with 9 ng of injected digest.     

Selective isolation of C-terminal peptides from proteolytic mixtures of proteins by affinity chromatography on immobilized anhydrotrypsin and anhydrochymotrypsin

Anhydrotrypsin in an immobilized form shows strong specific affinity for the peptides containing Arg, Lys, or S-aminoethyl-Cys at their C-termini. By taking advantage of this unique property of the adsorbent, we established an efficient chromatographic procedure useful for selective isolation of C-terminal peptides from protease digests of proteins. The utility was demonstrated in cases of a tryptic digest of Streptomyces subtilisin inhibitor in a reduced and S-carboxymethylated form (RCM-SSI) (with C-terminal Phe) and of a chymotryptic digest of α₁-antitrypsin (with C-terminal Lys): the C-terminal peptides were recovered as the breakthrough fraction of the chromatography in the former case and as the adsorptive fraction in the latter. Immobilized anhydrochymotrypsin was also useful for selective adsorption of the C-terminal peptide from the tryptic digest of RCM-SSI. It was further found that immobilized anhydrotrypsin exerts strong affinity even for human oxyhemoglobin and its α-chain (with C-terminal Arg), but not for the β-chain (with C-terminal His) and hemoglobin-haptoglobin complex. Thus the adsorbent may be applicable also to the isolation of macromolecular ligands and to the analysis of macromolecular interactions.     

Highly efficient enrichment of phosphopeptides by magnetic nanoparticles coated with zirconium phosphonate for phosphoproteome analysis

The location of phosphorylation plays a vital role for the elucidation of biological processes. The challenge of low stoichiometry of phosphoproteins and signal suppression of phosphopeptides by nonphosphopeptides in mass spectrometry (MS) analysis makes the selective enrichment of phosphopeptides prior to MS analysis necessary. Besides the immobilized metal affinity chromatography (IMAC) method, some affinity methods based on nanoparticles displayed a higher enrichment efficiency for phosphopeptides such as Fe(3)O(4)/TiO2 and Fe(3)O(4)/ZrO(2) nanoparticles. To further improve the selectivity and compatibility of the affinity methods, a novel strategy based on magnetic nanoparticles coated with zirconium phosphonate for the enrichment of phosphopeptides has been developed in this study. Under optimized experimental conditions, 1 x 10(-9) M phosphopeptides in 50 microL tryptic digest of beta-casein could be enriched and identified successfully. Reliable results were also obtained for 1 x 10(-8) M phosphopeptides in 50 microL tryptic digest of beta-casein in the presence of nonphosphopeptides from a tryptic digest of bovine serum albumin (BSA) over 20 times in concentration. The performance of nanoparticles for use in a real sample was further demonstrated by employing the strong cation-exchange chromatography (SCX) fraction of a tryptic digest of a protein extract from Chang liver cells as a model sample. Experimental results show that the nanoparticles can be easily and effectively used for enrichment of phosphopeptides in low concentration. Most importantly, our approach is more compatible with commonly used SCX strategies than Fe(3+)-IMAC. The proposed method thus has great potential for future studies of large-scale phosphoproteomes.     

A New Approach to High Sensitivity Liquid Chromatography-Mass Spectrometry of Peptides using Nanoflow Solvent Assisted Inlet Ionization

Liquid chromatography (LC) solvent assisted inlet ionization (SAII) mass spectrometry (MS) was previously reported to give good chromatographic resolution and MS detection injecting 66 ng of a BSA tryptic digest. In analogy to nano-electrospray ionization (nESI), we extend SAII LC/MS to nano-SAII (nSAII) operating at nL min^–1 flow rates and demonstrate good quality ion chromatograms and mass spectra from injection of as little as 0.7 ng of BSA digest onto a capillary LC column. Data dependent fragmentation is demonstrated for injection of 7 ng of a BSA digest. This method has advantages over nESI in ease of use and low cost as it requires no voltage and is operational without the necessity of connectors or fragile nESI emitters, although similar constricted tips can be helpful in nSAII to stabilize the signal at low nanoliter flow. At a flow rate of 0.8 μL min^–1, the only requirement for nSAII is that the exit-end of the capillary LC column be adjusted near the aperture of the heated inlet of the mass spectrometer.     

电感耦合等离子体质谱测试硫化物中的微量元素

采用多种酸组合消解方法消解硫化物样品,最终选取A:HNO3密闭消解法;B:HCI+HF与HNO3两步消解法对硫化物标准物质GBW07267(黄铁矿)、GBW07268(黄铜矿),GBW07270(闪锌矿)及硫化物标准物质WMS-1a(块状硫化物)样品进行消解,采用ICP-MS测试了39种微量元素.结果表明,HNO3密闭...     

Purification of DNA-derived deoxynucleotides from leukocytes involving nuclease elution of an ion-exchange column

A method has been developed in which the DNA of leukocytes (as the buffy coat from blood) is isolated in the form of its constituent deoxynucleotides. The steps in this method are as follows: (1) lyse the leukocytes with sodium dodecyl sulfate (SDS) and enzymatically digest the proteins and RNA, (2) remove the SDS on a non-polar adsorbent (Bio-Beads SM-4) and then trap the DNA on a quaternary amine silica cartridge, (3) wash the column with 1 M NaCl-buffer, (4) digest the DNA on the column with staphylococcal nuclease and (5) elute the digested DNA with 0.5 M NaCl-buffer and digest it further with bovine spleen phosphodiesterase II to deoxynucleotide-3′-monophosphates. From a 40-μl sample of butty coat was obtained 126 ± 14 μg (two experiments, eight sample total) of deoxynucleotides. Reversed-phase high-performance liquid chromatography, which removed the added enzymes, showed only peaks for deoxynucleotides. For comparison, the amount of deoxynucleotides obtained from the leukocytes by an automated phenol extraction procedure was 101 ± 5.4 μg (one experiment in triplicate).     

Improved peptide mass fingerprinting matches via optimized sample preparation in MALDI mass spectrometry

Peptide mass fingerprinting (PMF) is a powerful technique in which experimentally measured m/z values of peptides resulting from a protein digest form the basis for a characteristic fingerprint of the intact protein. Due to its propensity to generate singly charged ions, along with its relative insensitivity to salts and buffers, matrix-assisted laser desorption and ionization (MALDI)-time-of-flight mass spectrometry (TOFMS) is the MS method of choice for PMF. The qualitative features of the mass spectrum can be selectively tuned by employing different methods to prepare the protein digest and matrix for MALDI-TOFMS. The selective tuning of MALDI mass spectra in order to optimize PMF is addressed here. Bovine serum albumin, carbonic anhydrase, cytochrome c, hemoglobin alpha- and beta-chain, and myoglobin were digested with trypsin and then analyzed by MALDI-TOFMS. 2,5-dihydroxybenzoic acid (DHB) and alpha-cyano-4-hydroxycinnamic acid (CHCA) were prepared using six different sample preparation methods: dried droplet, application of protein digest on MALDI plate followed by addition of matrix, dried droplet with vacuum drying, overlayer, sandwich, and dried droplet with heating. Improved results were obtained for the matrix alpha-cyano-4-hydroxycinnamic acid using a modification of the died droplet method in which the MALDI plate was heated to 80 °C prior to matrix application, which is supported by observations from scanning electron microscopy. Although each protein was found to have a different optimum sample preparation method for PMF, in general higher sequence coverage for PMF was obtained using DHB. The best PMF results were obtained when all of the mass spectral data for a particular protein digest was convolved together.     

Development and evaluation of on-line nanoliter flow analysis of protein digests by pneumatic-splitter electrospray liquid chromatography mass spectrometry

We have developed a novel approach to characterize protein digests by pneumatic-splitter electrospray ionization liquid chromatography mass spectrometry (PSESI-LCMS). This technique involves an interfacing of a pneumatic splitter that can dynamically generate and control a steady split flow rate of nanoliters per minute. An average PSESI-LCMS analysis, depending on the desired split ratio, consumes less than 200 femtomoles of sample. A tryptic digest map of a glycoprotein, fetuin, and a Lys-C digest map of a recombinant neurotrophin protein, neurotrophin-3, are reported here for this investigation.