Improved sensitivity for insulin in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry by premixing alpha-cyano-4-hydroxycinnamic acid matrix with transferrin

This report describes an enhancement of the signal intensities of proteins and peptides in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). When alpha-cyano-4-hydroxycinnamic acid (CHCA) premixed with human transferrin (Tf) was used as a matrix, the signal intensity of insulin was amplified to more than ten times that of the respective control in CHCA without Tf. The detection limit of insulin was 0.39 fmol on-probe in the presence of Tf, while it was 6.3 fmol in the absence of Tf. The signal intensity of insulin was also enhanced when the CHCA matrix was premixed with proteins other than Tf (80 kDa), such as horse ferritin (20 kDa), bovine serum albumin (BSA, 66 kDa), or human immunoglobulin G (150 kDa). The optimum spectrum of insulin was obtained when the added amount of protein was in the range 0.26-0.62 pmol, regardless of the molecular weight of the added protein. Tf and BSA outperformed the other tested proteins, as determined by improvements in the resulting spectra. When the mass spectra of several peptides and proteins were recorded in the presence of Tf or BSA, the signal intensities of large peptides such as glucagon were enhanced, though those of smaller peptides were not enhanced. In addition, the signal enhancement achieved with Tf and BSA was more pronounced for the proteins, including cytochrome C, than for the large peptides. This enhancement effect could be applied to improve the sensitivity of MALDI-TOFMS to large peptides and proteins.     

Characterization of γ-carboxylated tryptic peptides by collision-induced dissociation and electron transfer dissociation mass spectrometry

Vitamin K-dependent carboxylation of glutamic acid (Glu) residues into γ-carboxyglutamic acid (Gla) is a post-translational modification essential for normal protein activity of, for example, proteins involved in the blood coagulation system. These proteins may contain as many as 12 sites for γ-carboxylation within a protein sequence of 45 amino acid residues. In the biopharmaceutical industry, powerful analytical techniques are required for identification and localization of modified sites. We here present comparatively easy and rapid methods for studies of Gla-containing proteins using recent technology. The performances of two mass spectrometric fragmentation techniques, collision-induced dissociation (CID) and electron transfer dissociation (ETD), were evaluated with respect to γ-carboxylated peptides, applying on-line LC-ion trap MS. ETD MS has so far not been reported for Gla-containing peptides and the applicability of CID for heavily γ-carboxylated proteins has not been evaluated. The anticoagulant protein, protein C, containing nine Gla-sites, was chosen as a model protein. After tryptic digestion, three peptides containing Gla-residues were detected by MS; a 1.2 kDa fragment containing two Gla-residues, a 4.5 kDa peptide containing seven residues and also the 5.6 kDa tryptic peptides containing all nine Gla-residues. Regarding the shortest peptide, both CID and ETD provided extensive peptide sequencing. For the larger peptides, fragmentation by CID resulted in loss of the 44 Da CO(2)-group, while little additional fragmentation of the peptide chain was observed. In contrast, ETD resulted in comprehensive fragmentation of the peptide backbone. The study demonstrates that the combination of both techniques would be beneficial and complementary for investigation of γ-carboxylated proteins and peptides.     

Improved disc SDS‐PAGE for extraction of low molecular weight proteins from serum

The low molecular weight proteins can provide a lot of valuable information of biomarkers. To study these proteins, the high abundance and high molecular weight proteins must be removed prior to analysis. In this work, a simple and inexpensive disc SDS‐PAGE to extract low molecular weight proteins from human serum and cutoff proteins larger than 30 kDa was developed. Some experimental conditions were examined. The experimental results obtained by plate SDS‐PAGE and MALDI‐TOF MS showed that the molecular weight of extracted proteins was about in the range from 0.3 to 28 kDa. Some experiments, including precipitation of proteins in organic solvents, SPE and cytochrome C test, were carried out and the experimental results demonstrated successful recovery of proteins/peptides with molecular weight from several hundreds of dalton to about 30 kDa. The experimental results obtained by plate SDS‐PAGE indicated the repeatability was satisfactory.     

Peptidomics: the comprehensive analysis of peptides in complex biological mixtures

Progress in the sequencing of genomes has resulted in an increasing demand for a functional analysis of gene products in order to understand the underlying physiology. Proteomics has established itself as a highly valuable technology for producing functionally related data in an unparalleled fashion, but is methodologically restricted to the analysis of proteins with higher molecular masses (>10 kDa). The development of a technology which covers peptides with low molecular weight and small proteins (0.5 to 15 kDa) was necessary, since peptides, amongst them families of hormones, cytokines and growth factors, play a central role in many biological processes. To summarise the technologies used for this approach the term "peptidomics" is introduced. In this article, we present the rationale and first results of a novel, universal peptide display approach for the analysis and visualisation of peptides and small proteins from biological samples. Special attention is given to samples derived from extracellular fluids such as blood plasma and cerebrospinal fluid. Additionally, a high throughput identification procedure for the analysis of peptides in their native and processed molecular form is outlined.     

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

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

Selective extraction of low molecular weight proteins by mesoporous silica particles with modified internal and external surfaces

A new mesoporous silica material with modified external and internal surfaces (alkyl diol–vinyl-SiO₂) was prepared and applied to selectively extract low molecular weight (LMW) proteins and peptides (less than 11 kDa) from biological samples. X-ray diffraction, N₂ adsorption, transmission electron microscopy, and Fourier transform infrared spectroscopy were used to characterize the alkyl diol–vinyl-SiO₂. This material was used to extract LMW proteins (peptides) from standard proteins, a mixture of peptides, and crude human plasma. We compared the amounts of different proteins adsorbed by alkyl diol–vinyl-SiO₂ and by vinyl-SiO₂ (vinyl group on the internal surface of the mesoporous silica material). The desorption recovery of alkyl diol–vinyl-SiO₂ by different eluents was also investigated by using a standard protein (insulin) as a model LMW protein. The material could efficiently extract LMW proteins and peptides from a mixture of standard proteins and crude human plasma with good extraction efficiency and desorption recovery. Furthermore, the alkyl diol–vinyl-SiO₂ had much better extraction selectivity for LMW proteins and peptides than a commercial C₁₈ solid phase extraction material.     

Rapid capillary electrophoresis time-of-flight mass spectrometry separations of peptides and proteins using a monoquaternarized piperazine compound (M7C4I) for capillary coatings

A monoquaternarized piperazine, 1-(4-iodobutyl) 4-aza-1-azoniabicyclo[2,2,2] octane iodide (M7C4I), has been evaluated as a surface derivatization reagent for CE in combination with TOF MS for the analysis of proteins, peptides, and protein digests. The M7C4I piperazine, at alkaline pH, forms a covalent bond via alkylation of the ionized silanols producing a cationic surface with a highly stable and reversed EOF. The obtained surface yields rapid separations (less than 5 min) of peptides and proteins at acidic pH with high separation efficiencies (up to 1.1 x 10(6) plates/m for peptides and up to 1.8 x 10(6) plates/m for proteins) and no observed bleeding of the coating reagent into the mass spectrometer. The simplicity of the coating procedure also enables fast (2 min) regeneration of the surface, if necessary. This is useful in the analysis of complex samples in order to prevent possible memory effects. The potential of using M7C4I-coated capillaries for MS analysis of complex samples is demonstrated by the separation of peptides, proteins, and protein digests. Even more, the spectacular thing in which large intact proteins with molecular masses over 0.5 MDa could be separated. The coating showed good ability to handle these large proteins with high efficiency and retained peak shape as demonstrated by separation of IgG(1) (150 kDa) and thyroglobulin (669 kDa).     

Analysis of the human urine endogenous peptides by nanoparticle extraction and mass spectrometry identification

Peptides in urine are excreted by kidney from the blood and tissues, which are composed of a large amount of hormones, cytokines, regulatory factors and the metabolized fragments of proteins. The peptide distribution in urine will reflect the physiological and pathophysiological processes in body. In past, limited information was reported about the composition of the peptides in urine. One possible reason is that the peptides in urine are fairly low abundant and there are high concentrations of salts and organic metabolites in the urine. In this report, we extracted the peptides from human urine by highly ordered mesoporous silica particles with the pore size of 2 nm, which will exclude the high molecular weight proteins over 12 kDa. The extracted peptides were then separated into fractions according to their molecular weight by size exclusion chromatography. Each of the fractions was further analyzed by MALDI-TOF MS and μRPLC–MS/MS. Totally, 193 peptides were identified by two-dimensional SEC/μRPLC–MS/MS analysis. By analyzing the progenitor protein of the peptides; we found that two-thirds of the proteins differed from the reported urine proteome database, and the high abundant proteins in urine proteome were less detected in the urine peptidome. The developed extraction and separation methods were efficient for the profiling of the endogenous peptides in human urine. The peptidome in human urine was complementary to the human urinary proteome and may provide an emerging field for biomarker discovery.     

Improved selection of LMW over HMW proteins from human plasma by mesoporous silica particles with external modification

Selective extraction of low molecular weight (LMW) proteins and peptides from complex biological samples plays an important role in the discovery of useful biomarkers and signaling molecules. Various methods, such as solid-phase extraction (SPE), ultrafiltration, and size-exclusion chromatography have been developed for such extraction purpose. In this study, we present, to our knowledge, the first demonstration of alkyl-diol@SiO₂ mesoporous material MCM-41 (alkyl-diol group on the external surface of mesoporous material) for selective extraction of LMW proteins and peptides from complex biological samples. The adsorption kinetics of LMW proteins, the influence of pH on adsorption and the desorption recovery by different elution solvents were investigated by using standard proteins as model samples. It was demonstrated that the modification of alkyl-diol group on the external surface could efficiently decrease the adsorption of HMW protein and increase the desorption recovery of LMW protein. Furthermore, the mesoporous materials were applied to selectively extract LMW proteins and peptides (<10 kDa) from crude human plasma. And the modified MCM-41 material had much better extraction selectivity and efficiency for LMW proteins and peptides than unmodified one.     

MALDI imaging and profiling MS of higher mass proteins from tissue

MALDI imaging and profiling mass spectrometry of proteins typically leads to the detection of a large number of peptides and small proteins but is much less successful for larger proteins: most ion signals correspond to proteins of m/z < 25,000. This is a severe limitation as many proteins, including cytokines, growth factors, enzymes, and receptors have molecular weights exceeding 25 kDa. The detector technology typically used for protein imaging, a microchannel plate, is not well suited to the detection of high m/z ions and is prone to detector saturation when analyzing complex mixtures. Here we report increased sensitivity for higher mass proteins by using the CovalX high mass HM1 detector (Zurich, Switzerland), which has been specifically designed for the detection of high mass ions and which is much less prone to detector saturation. The results demonstrate that a range of different sample preparation strategies enable higher mass proteins to be analyzed if the detector technology maintains high detection efficiency throughout the mass range. The detector enables proteins up to 70 kDa to be imaged, and proteins up to 110 kDa to be detected, directly from tissue, and indicates new directions by which the mass range amenable to MALDI imaging MS and MALDI profiling MS may be extended.     

A proteomic view on the developmental transfer of homologous 30 kDa lipoproteins from peripheral fat body to perivisceral fat body via hemolymph in silkworm, Bombyx mori

Background

A group of abundant proteins of ~30 kDa is synthesized in silkworm larval peripheral fat body (PPFB) tissues and transported into the open circulatory system (hemolymph) in a time-depended fashion to be eventually stored as granules in the pupal perivisceral fat body (PVFB) tissues for adult development during the non-feeding stage. These proteins have been shown to act anti-apoptotic besides being assigned roles in embryogenesis and defense. However, detailed protein structural information for individual PPFB and PVFB tissues during larval and pupal developmental stages is still missing. Gel electrophoresis and chromatography were used to separate the 30 kDa proteins from both PPFB and PVFB as well as hemolymph total proteomes. Mass spectrometry (MS) was employed to elucidate individual protein sequences. Furthermore, 30 kDa proteins were purified and biochemically characterized.

Results

One- and two-dimensional gel electrophoresis (1/2D-PAGE) was used to visualize the relative changes of abundance of the 30 kDa proteins in PPFB and PVFB as well as hemolymph from day 1 of V instar larval stage to day 6 of pupal stage. Their concentrations were markedly increased in hemolymph and PVFB up to the first two days of pupal development and these proteins were consumed during development of the adult insect. Typically, three protein bands were observed (~29, 30, 31 kDa) in 1D-PAGE, which were subjected to MS-based protein identification along with spots excised from 2D-gels run for those proteomes. Gas phase fragmentation was used to generate peptide sequence information, which was matched to the available nucleotide data pool of more than ten highly homologous insect 30 kDa lipoproteins. Phylogenetic and similarity analyses of those sequences were performed to assist in the assignment of experimentally identified peptides to known sequences. Lipoproteins LP1 to LP5 and L301/302 could be matched to peptides extracted from all bands suggesting the presence of full length and truncated or modified protein forms in all of them. The individual variants could not be easily separated by classical means of purification such as 2D-PAGE because of their high similarity. They even seemed to aggregate as was indicated by native gel electrophoresis. Multistep chromatographic procedures eventually allowed purification of an LP3-like protein. The protein responded to lipoprotein-specific staining.

Conclusions

In B. mori larvae and pupae, 30 kDa lipoproteins LP1 to LP5 and L301/302 were detected in PPFB and PVFB tissue as well as in hemolymph. The concentration of these proteins changed progressively during development from their synthesis in PPFB, transport in hemolymph to storage in PVFB. While the 30 kDa proteins could be reproducibly separated in three bands electrophoretically, the exact nature of the individual protein forms present in those bands remained partially ambiguous. The amino acid sequences of all known 30 kDa proteins showed very high homology. High-resolution separation techniques will be necessary before MS and other structural analysis can shed more light on the complexity of the 30 kDa subproteome in B. mori. A first attempt to that end allowed isolation of a B. mori LP3-like protein, the complete structure, properties and function of which will now be elucidated in detail.     

自建电喷雾解吸电离源（DESI）在大分子分析中的应用及离子化机理探索

自建了简易的电喷雾解吸电离源(DESI),优化了DESI源喷口的位置和角度,并将其用于常见多肽和蛋白质的分析。多肽和小质量蛋白质(<20 kDa)可以容易地从表面解吸电离,生成清晰的质谱。而牛血清白蛋白(66.4 kDa)不能产生清晰的多电荷分布的质谱,说明当前DESI源的设计可能存在一个电离的分子量上限。通过比较不同的实验条件并对比ESI-MS,发现溶剂分子的挥发过程对电荷分布以及峰宽均有显著影响,可能是由于ESI更软引起。载样表面的性质对DESI-MS的信号强度有较大影响。金表面的自组装单分子膜(SAM)相对于纯金表面有较好的绝缘性,并有助于产生较强信号,说明来自表面的电子转移(电中和)是电喷雾解吸电离过程中的一个重要因素。该文的研究有助于对DESI-MS的实验条件和载样表面的选择,同时增进了对电喷雾解吸电离机理的了解。     

Integrated protein analysis platform based on column switch recycling size exclusion chromatography,microenzymatic reactor and μRPLC–ESI-MS/MS

An integrated platform with the combination of proteins and peptides separation was established via the unit of on-line proteins digestion, by which proteins were in sequence separated by column switch recycling size exclusion chromatography (csrSEC), on-line digested by an immobilized trypsin microreactor, trapped and desalted by two parallel C8 precolumns, separated by μRPLC with the linear gradient of organic modifier concentration, and identified by ESI-MS/MS. A 6-protein mixture, with Mr ranging from 10 kDa to 80 kDa, was used to evaluate the performance of the integrated platform, and all proteins were identified with sequence coverage over 5.67%. Our experimental results demonstrate that such an integrated platform is of advantages such as good time compatibility, high peak capacity, and facile automation, which might be a promising approach for proteome study.     

Comparative characteristics of water-soluble proteins from <Emphasis Type="Italic">Larix sibirica</Emphasis>, <Emphasis Type="Italic">Picea obovata</Emphasis>, and <Emphasis Type="Italic">Abies sibirica</Emphasis> bud meristem

The amount of water-soluble proteins in bud meristem of Larix sibirica L., Picea obovata L., and Abies sibirica L. was shown to increase by 2-3 times in autumn during development of low-temperature resistance. The fractional composition of water-soluble proteins of the studied species and the amino-acid composition of groups of water-soluble proteins with different molecular weight (MW) were similar. Nitrogen accumulated as aspartic acid, glycine, and alanine in high- and medium-molecular-weight proteins. The peptides (MW < 5 kDa) typically had a high content of hydrophobic proline and hydrophilic tyrosine.     

Probing high order structure of proteins by fast-atom bombardment mass spectrometry

During the past decade, numerous investigations have demonstrated that the rate at which amide hydrogens located at peptide linkages undergo isotopic exchange is a sensitive probe of the high order structure and dynamics of proteins. The present investigation demonstrates that microbore high-performance liquid chromatography (HPLC) continuous-flow fast-atom bombardment mass spectrometry (FABMS) can be used to accurately quantify deuterium located at peptide linkages in short segments of large proteins. This result is important because it demonstrates the feasibility of using mass spectrometry as a tool for studying the high order structure and dynamics of large proteins. Following a period of deuterium exchange-in, a protein was placed into slow-exchange conditions and fragmented into peptides with pepsin. The digest was analyzed by continuous-flow HPLC FABMS to determine the molecular weights of the peptides, from which the number of deuterons located at the peptide linkages could be deduced. The HPLC step was used both to fractionate the peptides according to their hydrophobicities and to remove through back-exchange all deuterium except that located at peptide amide linkages. This approach has been applied to α-crystallin, a lens protein composed of two gene products with monomer molecular weights of 20 kDa and an aggregate molecular weight approaching 1000 kDa. Results from this study show that some of the peptide amide hydrogens in αA-crystallin exchange very rapidly (k > 10 h^?1) while others exchange very slowly (k < 10^?3 h^?1). The ability not only to detect that a conformational change has occurred, but also to identify the specific regions within the protein where the change occurred, was demonstrated by measuring changes in the exchange rates within these regions as the deuterium exchange-in temperature was increased from 10 to 80 ° C.     

Antigiardial activity of glycoproteins and glycopeptides from Ziziphus honey

Natural honey contains an array of glycoproteins, proteoglycans and glycopeptides. Size-exclusion chromatography fractionated Ziziphus honey proteins into five peaks with molecular masses in the range from 10 to >200 kDa. The fractionated proteins exhibited in vitro activities against Giardia lamblia with IC₅₀ values ≤ 25 μg/mL. Results indicated that honey proteins were more active as antiprotozoal agents than metronidazole. This study indicated the potential of honey proteins and peptides as novel antigiardial agents.     

Top-down analysis of 30–80 kDa proteins by electron transfer dissociation time-of-flight mass spectrometry

Electron transfer dissociation (ETD)-based top-down mass spectrometry (MS) is the method of choice for in-depth structure characterization of large peptides, small- and medium-sized proteins, and non-covalent protein complexes. Here, we describe the performance of this approach for structural analysis of intact proteins as large as the 80 kDa serotransferrin. Current time-of-flight (TOF) MS technologies ensure adequate resolution and mass accuracy to simultaneously analyze intact 30–80 kDa protein ions and the complex mixture of their ETD product ions. Here, we show that ETD TOF MS is efficient and may provide extensive sequence information for unfolded and highly charged (around 1 charge/kDa) proteins of ～30 kDa and structural motifs embedded in larger proteins. Sequence regions protected by disulfide bonds within intact non-reduced proteins oftentimes remain uncharacterized due to the low efficiency of their fragmentation by ETD. For serotransferrin, reduction of S–S bonds leads to significantly varied ETD fragmentation pattern with higher sequence coverage of N- and C-terminal regions, providing a complementary structural information to top-down analysis of its oxidized form.

Direct high-resolution peptide and protein analysis by desorption electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

We report the first coupling of a desorption electrospray ionization (DESI) ion source to Fourier transform ion cyclotron resonance mass spectrometry (ESI-FT-ICR-MS) for high-resolution protein analysis. The DESI FT-ICR-MS source design is described in detail along with preliminary data obtained on peptides and proteins ranging from 1 to 5.7 kDa.     

Sequencing membrane proteins by tandem mass spectrometry

Tandem mass spectrometry (MS/MS) is an attractive technique for sequencing membrane proteins because it can be applied to peptides in mixtures that are difficult to separate chromatographically. To evaluate the suitability of MS/MS sequencing for membrane proteins and to develop protocols for the preparation of the cleaved peptides, we employed the well characterized apoproteins of bacteriorhodopsin and bovine rhodopsin, i.e. bacterioopsin and opsin, respectively. Without separation, nine out of ten peptides resulting from cyanogen bromide cleavage of bacterioopsin were detected by fast atom bombardment MS, the single undetected fragment being a tetrapeptide that was presumably hidden in the low-m/z matrix background. Furthermore, MS/MS was used to confirm the sequence of all the peptides detected with m/z values below 3.5 kDa (40% of the protein). Bovine opsin was analyzed in a similar fashion. Tandem MS/MS has thus allowed the sequencing of substantial portions of two integral membrane proteins by the analysis of unseparated peptide mixtures, demonstrating for the first time that this technique can obviate some of the most serious difficulties associated with sequencing membrane proteins, namely the difficult-to-achieve separation of the ‘sticky’ peptide fragments.     

Human cerebrospinal fluid peptidomics

Cerebrospinal fluid (CSF) has frequently been extensively studied to explore several different central nervous system (CNS) disorders because it contains proteins, enzymes, hormones, neuropeptides and neurotransmitters that play critical regulatory roles in many different physiological processes. Individual neuropeptidergic systems in CSF have been studied. In theory, peptidomics offers a bird's-eye, comprehensive and systems-level approach to analyze all of the peptidergic systems that have been expressed in CSF at any given time. In this study, low molecular mass (M(r) < 5 kDa) peptides were isolated by ultrafiltration. The isolated peptides, with or without trypsin digestion, were preferentially enriched with a solid-phase extraction cartridge, and the peptides were separated with capillary liquid chromatography and analyzed with on-line quadrupole time-of-flight mass spectrometry (MS). In this proof-of-principle study, the 20 representative MS-characterized peptides were shown to be derived from 12 proteins, among which four proteins, amyloid-like protein 1, secretogranin I, granin-like neuroendocrine peptide precursor and neurosecretory protein VGF, have been shown elsewhere to be either associated with CNS disorders or to play a central role in the CNS. The long-term goals of this peptidomics study are to monitor the changes (amount; modifications) of CSF peptides, clarify the aberrant processing of large intact protein precursors, elucidate the molecular mechanisms of CNS disorders and find biomarkers. This analytical method is effective for the analysis of the human lumbar CSF peptidome.