Efficient Tandem LysC/Trypsin Digestion in Detergent Conditions

All shotgun proteomics experiments rely on efficient proteolysis steps for sensitive peptide/protein identification and quantification. Previous reports suggest that the sequential tandem LysC/trypsin digest yields higher recovery of fully tryptic peptides than single‐tryptic proteolysis. Based on the previous studies, it is assumed that the advantageous effect of tandem proteolysis requires a high sample denaturation state for the initial LysC digest. Therefore, to date, all systematic assessments of LysC/trypsin proteolysis are done in chaotropic environments such as urea. Here, sole trypsin is compared with LysC/trypsin and it is shown that tandem digestion can be carried with high efficiency in Mass Spectrometry‐compatible detergents, thereby resulting in higher quantitative yields of fully cleaved peptides. It is further demonstrated that higher cleavage efficiency of tandem digests has a positive impact on absolute protein quantification using intensity‐based absolute quantification (iBAQ) values. The results of the examination of divergent urea tandem conditions imply that beneficial effects of the initial LysC digest do not depend on the sample denaturation state, but, are mainly caused by different target specificities of LysC and trypsin. The observed detergent compatibility enables tandem digestion schemes to be implemented in efficient cellular solubilization proteomics procedures without the need for buffer exchange to chaotropic environments.     

The utility of proteases in proteomics,from sequence profiling to structure and function analysis

In mass spectrometry (MS)-based bottom-up proteomics, protease digestion plays an essential role in profiling both proteome sequences and post-translational modifications (PTMs). Trypsin is the gold standard in digesting intact proteins into small-size peptides, which are more suitable for high-performance liquid chromatography (HPLC) separation and tandem MS (MS/MS) characterization. However, protein sequences lacking Lys and Arg cannot be cleaved by trypsin and may be missed in conventional proteomic analysis. Proteases with cleavage sites complementary to trypsin are widely applied in proteomic analysis to greatly improve the coverage of proteome sequences and PTM sites. In this review, we survey the common and newly emerging proteases used in proteomics analysis mainly in the last 5 years, focusing on their unique cleavage features and specific proteomics applications such as missing protein characterization, new PTM discovery, and de novo sequencing. In addition, we summarize the applications of proteases in structural proteomics and protein function analysis in recent years. Finally, we discuss the future development directions of new proteases and applications in proteomics.     

Improve the coverage for the analysis of phosphoproteome of HeLa cells by a tandem digestion approach

Complete coverage of all phosphorylation sites in a proteome is the ultimate goal for large-scale phosphoproteome analysis. However, only making use of one protease trypsin for protein digestion cannot cover all phosphorylation sites, because not all tryptic phosphopeptides are detectable in MS. To further increase the phosphoproteomics coverage of HeLa cells, we proposed a tandem digestion approach by using two different proteases. By combining the data set of the first Glu-C digestion and the second trypsin digestion, the tandem digestion approach resulted in the identification of 8062 unique phosphopeptides and 8507 phosphorylation sites in HeLa cells. The conventional trypsin digestion approach resulted in the identification of 3891 unique phosphopeptides and 4647 phosphorylation sites. It was found that the phosphorylation sites identified from the above two approaches were highly complementary. By combining above two data sets, in total we identified 10899 unique phosphopeptides and 11262 phosphorylation sites, corresponding to 3437 unique phosphoproteins with FDR < 1% at peptide level. We also compared the kinase motifs extracted from trypsin, Glu-C, or a second trypsin digestion data sets. It was observed that basophilic motifs were more frequently found in the trypsin and the second trypsin digestion data sets, and the acidic motifs were more frequently found in the Glu-C digestion data set. These results demonstrated that our tandem digestion approach is a good complement to the conventional trypsin digestion approach for improving the phosphoproteomics analysis coverage of HeLa cells.     

Mass spectrometrical analysis of the processed metastasis-inducing <Emphasis Type="Italic">anterior gradient protein 2 homolog</Emphasis> reveals 100% sequence coverage

Anterior gradient protein 2 homolog is a metastasis-inducing protein in a rat model of rat breast cancer and prognostic for outcome in hormonally treated breast cancer patients. Carrying out protein profiling in several mammalian cells and tissues, we detected this protein (synonym: secreted cement gland protein XAG-2 homolog) that was originally described in toad skin, in human bronchial epithelia. Tissues obtained from biopsies were homogenised and extracted proteins were run on two-dimensional gel electrophoresis. Following in-gel digestion with proteases trypsin, AspN, LysC and chymotrypsin, mass spectrometrical analysis was carried out by MALDI-TOF/TOF. The use of MS following multi-enzyme digestion of the protein resulted into 100% sequence coverage. MS/MS analysis enabled sequencing of 87% of the protein structure. This percentage does not include the signal peptide that was not observed in our protein due to processing. No posttranslational modifications were detectable and no sequence conflicts were observed. Complete analysis, unambiguous identification and characterisation of this biologically important protein could be shown, which is relevant for the definition of a marker protein that has been described so far by immunochemical methods only. Complete analysis is of importance as it forms the basis for all future work on this protein and, moreover, may serve as an analytical tool for further studies.     

The Origin of Trypsin: Evidence for Multiple Gene Duplications in Trypsins

The trypsin family of serine proteases is one of the most studied protein families, with a wealth of amino acid sequence information available in public databases. Since trypsin-like enzymes are widely distributed in living organisms in nature, likely evolutionary scenarios have been proposed. A novel methodology for Fourier transformation of biological sequences (FOTOBIS) is presented. The methodology is well suited for the identification of the size and extent of short repeats in protein sequences. In the present paper the trypsin family of enzymes is analyzed with FOTOBIS and strong evidence for tandem gene duplication is found. A likely evolutionary path for the development of present-day trypsins involved an intrinsic extensive tandem gene duplication of a small DNA fragment of 15–18 nucleotides, corresponding to five or six amino acids. This ancestral trypsin gene was subsequently duplicated, leading to the earliest version of a full-sized trypsin, from which the contemporary trypsins have developed. Received: 22 November 1997 / Accepted: 26 January 1998     

Characterization of serine proteases of Lumbriculus variegatus and their role in regeneration

Serine proteases, ubiquitous enzymes known to function in digestion and immune protection in both vertebrates and invertebrates and implicated in regeneration in some species, were investigated in the California blackworm, Lumbriculus variegatus. Several serine proteases, rather than a single enzyme with broad specificity, were present in tissue extracts from the worms. Extracts were treated with a fluorescein‐labeled peptide chloromethyl ketone that specifically binds to trypsin/thrombin‐like proteases. Denaturing gel electrophoresis of labeled extracts showed several serine proteases with their molecular weight ranging 28,000–38,000 daltons. The trypsin/thrombin‐like activity was localized, using the fluorescein‐conjugated reagent, to the pharynx and digestive tract of L. variegatus. Movement of cells labeled by the reagent into regenerating tissues suggests that some differentiated endodermal tissues were used for reformation of digestive structures during regeneration in L. variegatus. The types of serine proteases in the extracts were further characterized by inhibitor studies. Presence of plasmin‐like activity was indicated by degradation of fibrin by tissue homogenates from the worms and the inhibitory effect of aprotinin on enzymes in these extracts. The ability of L. variegatus extracts to generate clots when incubated with rabbit plasma and partial inhibition of extract activity by phenylmethylsulfonyl fluoride and hirudin indicated presence of thrombin‐like activity. Consistent with the detection of trypsin, chymotrypsin, and plasmin‐like enzymes in the extracts was partial inhibition of L. variegatus serine protease activity by aminoethyl benzenesulfonyl fluoride and soybean trypsin inhibitor. Selective inhibition of chymotrypsin‐like activity by N‐tosyl‐l ‐phenylalanine chloromethyl ketone and chymostatin as well as trypsin‐like activity by N‐tosyl‐l ‐lysine chloromethyl ketone was observed. A potential role during regeneration for serine proteases is suggested by blockage of formation of head and tail structures by aminoethyl benzenesulfonyl fluoride, an inhibitor of these proteases.     

Evaluation of the possible proteomic application of trypsin from Streptomyces griseus

Trypsin (EC 3.4.21.4) is the protease of choice for proteome analysis using mass spectrometry of peptides in sample digests. In this work, trypsin from Streptomyces griseus (SGT) was purified to homogeneity from pronase. The enzyme was evaluated in in-gel digestion of protein standards followed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) analyses of the digests. We recognized a remarkable cleavage performance of SGT. The number of produced and matching tryptic peptides was higher than in the case of commonly used bovine trypsin (BT) and allowed us to obtain higher identification scores in database searches. Interestingly, SGT was found to also generate nonspecific peptides whose sequencing by MALDI-TOF/TOF tandem mass spectrometry (MS/MS) revealed a partial F-X, Y-X, and W-X cleavage specificity. To suppress autolysis, either arginine or arginine plus lysine residues in SGT were modified by chemical reagents. In consequence, the autolytic pattern of SGT was reduced significantly, but specific activity dropped dramatically. As demonstrated by relative quantification of peptides at different times, SGT is more stable at 37 °C than is its bovine counterpart. We conclude that SGT represents a convenient alternative for proteomic applications involving protein digestion. Moreover, parallel digestions of sample aliquots by SGT and BT provide the possibility of combining partially different results (unique matching peptides) to improve protein identification.     

Effect of inhibitory activity of mutation at reaction site P4 of the Streptomyces subtilisin inhibitor, SSI

The protein Streptomyces subtilisin inhibitor, SSI, efficiently inhibits a bacterial serine protease, subtilisin BPN'. We recently demonstrated that functional change in SSI was possible simply by replacing the amino acid residue at the reactive P1 site (methionine 73) of SSI. The present paper reports the additional effect of replacing methionine 70 at the P4 site of SSI (Lys73) on inhibitory activity toward two types of serine proteases, trypsin (or lysyl endopeptidase) and subtilisin BPN'. Conversion of methionine 70 at the P4 site of SSI(Lys73) to glycine or alanine resulted in increased inhibitory activity toward trypsin and lysyl endopeptidase, while replacement with phenylalanine weakened the inhibitory activity toward trypsin. This suggests that steric hindrance at the P4 site of SSI(Lys73) is an obstacle for its binding with trypsin. In contrast, the same P4 replacements had hardly any effect on inhibitory activity toward subtilisin BPN'. Thus the subsite structure of subtilisin BPN' is tolerant to these replacements. This contrast in the effect of P4 substitution might be due to the differences in the S4 subsite structures between the trypsin-like and the subtilisin-like proteases. These findings demonstrate the importance of considering structural complementarity, not only at the main reactive site but also at subsites of a protease, when designing stronger inhibitors.     

MALDI‐TOF and nESI Orbitrap MS/MS identify orthogonal parts of the phosphoproteome

Phosphorylation is a reversible posttranslational protein modification which plays a pivotal role in intracellular signaling. Despite extensive efforts, phosphorylation site mapping of proteomes is still incomplete motivating the exploration of alternative methods that complement existing workflows. In this study, we compared tandem mass spectrometry (MS/MS) on matrix assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) and nano‐electrospray ionization (nESI) Orbitrap instruments with respect to their ability to identify phosphopeptides from complex proteome digests. Phosphopeptides were enriched from tryptic digests of cell lines using Fe‐IMAC column chromatography and subjected to LC‐MS/MS analysis. We found that the two analytical workflows exhibited considerable orthogonality. For instance, MALDI‐TOF MS/MS favored the identification of phosphopeptides encompassing clear motif signatures for acidic residue directed kinases. The extent of orthogonality of the two LC‐MS/MS systems was comparable to that of using alternative proteases such as Asp‐N, Arg‐C, chymotrypsin, Glu‐C and Lys‐C on just one LC‐MS/MS instrument. Notably, MALDI‐TOF MS/MS identified an unexpectedly high number and percentage of phosphotyrosine sites (～20% of all sites), possibly as a direct consequence of more efficient ionization. The data clearly show that LC‐MALDI MS/MS can be a useful complement to LC‐nESI MS/MS for phosphoproteome mapping and particularly so for acidic and phosphotyrosine containing peptides.     

ICPLQuant – A software for non‐isobaric isotopic labeling proteomics

The main goal of many proteomics experiments is an accurate and rapid quantification and identification of regulated proteins in complex biological samples. The bottleneck in quantitative proteomics remains the availability of efficient software to evaluate and quantify the tremendous amount of mass spectral data acquired during a proteomics project. A new software suite, ICPLQuant, has been developed to accurately quantify isotope‐coded protein label (ICPL)‐labeled peptides on the MS level during LC‐MALDI and peptide mass fingerprint experiments. The tool is able to generate a list of differentially regulated peptide precursors for subsequent MS/MS experiments, minimizing time‐consuming acquisition and interpretation of MS/MS data. ICPLQuant is based on two independent units. Unit 1 performs ICPL multiplex detection and quantification and proposes peptides to be identified by MS/MS. Unit 2 combines MASCOT MS/MS protein identification with the quantitative data and produces a protein/peptide list with all the relevant information accessible for further data mining. The accuracy of quantification, selection of peptides for MS/MS‐identification and the automated output of a protein list of regulated proteins are demonstrated by the comparative analysis of four different mixtures of three proteins (Ovalbumin, Horseradish Peroxidase and Rabbit Albumin) spiked into the complex protein background of the DGPF Proteome Marker.     

Automated methods for improved protein identification by peptide mass fingerprinting

In order to maximize protein identification by peptide mass fingerprinting noise peaks must be removed from spectra and recalibration is often required. The preprocessing of the spectra before database searching is essential but is time-consuming. Nevertheless, the optimal database search parameters often vary over a batch of samples. For high-throughput protein identification, these factors should be set automatically, with no or little human intervention. In the present work automated batch filtering and recalibration using a statistical filter is described. The filter is combined with multiple data searches that are performed automatically. We show that, using several hundred protein digests, protein identification rates could be more than doubled, compared to standard database searching. Furthermore, automated large-scale in-gel digestion of proteins with endoproteinase LysC, and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis, followed by subsequent trypsin digestion and MALDI-TOF analysis were performed. Several proteins could be identified only after digestion with one of the enzymes, and some less significant protein identifications were confirmed after digestion with the other enzyme. The results indicate that identification of especially small and low-abundance proteins could be significantly improved after sequential digestions with two enzymes.     

S' subsite mapping of serine proteases based on fluorescence resonance energy transfer.

A microassay based on fluorescence resonance energy transfer has been developed to determine the S' specificity of serine proteases. The protease-catalyzed acyl transfer from a fluorescing acyl donor ester to a P'1/P'2 variable hexapeptide library of nucleophiles labeled with a fluorescence quencher leads to an internally quenched peptide product and a fluorescent hydrolysis product. The amount of fluorescence quenching allows one to draw conclusions about the interaction of the nucleophile at the S' sites of the protease. o-Aminobenzoic acid and 3-nitrotyrosine were used as an efficient donor-acceptor pair for the resonance energy transfer. The P'1/P'2 variable hexapeptide library with the general structure H-Xaa-Ala-Ala-Ala-Tyr(NO2)-Gly-OH and H-Ala-Xaa-Ala-Ala-Tyr(NO2)-Gly-OH, where Xaa represents Arg, Lys, Met, Phe, Ala, Gly, Ser, Gln and Glu, was prepared by solid-phase synthesis. Investigations of the S' specificity of trypsin, chymotrypsin and trypsin variants show that this assay is a fast and sensitive screening method for S' subsite mapping of serine proteases and is suitable for a high throughput screening. The assay might be useful for the development of restriction proteases and the estimation of yields in enzymatic peptide synthesis.     

An accurate proteomic quantification method: Fluorescence labeling absolute quantification (FLAQ) using multidimensional liquid chromatography and tandem mass spectrometry

A facile proteomic quantification method, fluorescent labeling absolute quantification (FLAQ), was developed. Instead of using MS for quantification, the FLAQ method is a chromatography-based quantification in combination with MS for identification. Multidimensional liquid chromatography (MDLC) with laser-induced fluorescence (LIF) detection with high accuracy and tandem MS system were employed for FLAQ. Several requirements should be met for fluorescent labeling in MS identification: Labeling completeness, minimum side-reactions, simple MS spectra, and no extra tandem MS fragmentations for structure elucidations. A fluorescence dye, 5-iodoacetamidofluorescein, was finally chosen to label proteins on all cysteine residues. The fluorescent dye was compatible with the process of the trypsin digestion and MALDI MS identification. Quantitative labeling was achieved with optimization of reacting conditions. A synthesized peptide and model proteins, BSA (35 cysteines), OVA (five cysteines), were used for verifying the completeness of labeling. Proteins were separated through MDLC and quantified based on fluorescent intensities, followed by MS identification. High accuracy (RSD% < 1.58) and wide linearity of quantification (1-10(5) ) were achieved by LIF detection. The limit of quantitation for the model protein was as low as 0.34 amol. Parts of proteins in human liver proteome were quantified and demonstrated using FLAQ.     

Epitope structure and binding affinity of single chain llama anti‐β‐amyloid antibodies revealed by proteolytic excision affinity‐mass spectrometry

ß‐Amyloid (Aß) immunotherapy has become a promising strategy for reducing the level of Aß in brain. New immunological approaches have been recently proposed for rapid, early diagnosis, and molecular treatment of neurodegenerative diseases related to Alzheimer's Disease (AD). The combination of proteolytic epitope excision and extraction and mass spectrometry using digestion with various proteases has been shown to be an efficient tool for the identification and molecular characterization of antigenic determinants. Here, we report the identification of the Aβ epitope recognized by the variable domain of single chain llama anti‐Aβ‐antibodies, termed Aβ‐nanobodies, that have been discovered in the blood of camelids and found to be promising candidates for immunotherapy of AD. The epitope recognized by two Aβ‐specific nanobodies was identified by proteolytic epitope extraction‐ and excision‐mass spectrometry using a series of proteases (trypsin, chymotrypsin, GluC‐protease, and LysC‐protease). Matrix‐assisted laser desorption ionization – mass spectrometric analysis of the affinity – elution fraction provided the epitope, Aβ(17–28), in the mid‐ to carboxy‐terminal domain of Aβ, which has been shown to exert an Aß‐fibril inhibiting effect. Affinity studies of the synthetic epitope confirmed that the Aβ(17–28) peptide is the minimal fragment that binds to the nanobodies. The interactions between the nanobodies and full length Aβ(1–40) or Aβ‐peptides containing or lacking the epitope sequence were further characterized by enzyme linked immunosorbent assay and bioaffinity analysis. Determinations of binding affinities between the Aβ‐nanobodies and Aβ(1–40) and the Aβ(17–28) epitope provided K_D values of approximately 150 and 700 nmol, respectively. Thus, the knowledge of the epitope may be highly useful for future studies of Aβ‐aggregation (oligomerization and fibril formation) and for designing new aggregation inhibitors. Copyright © 2012 John Wiley & Sons, Ltd.     

Limited acid hydrolysis as a means of fragmenting proteins isolated upon ProteinChip array surfaces

ProteinChip array technology enables protein purification, protein profiling, and biomarker discovery on a convenient biochip platform. Traditional proteomic approaches towards protein identification rely upon the generation of peptides through the use of specific proteases. However, for a variety of reasons, the digestion of proteins bound to planar arrays by specific proteases, such as trypsin, has proven to be difficult, at times providing little or no protein digestion at all. Additionally, should more than one protein be present on the array surface, the digestion product consists of peptides from different proteins, adding another dimension of complexity to database mining approaches. These factors have driven our group to explore alternative means of on-chip protein digestion. In this article, we describe an approach to generate peptide maps by limited acid hydrolysis. Depending upon the adsorbed protein, this method requires between 500 femtomole to 5 picomole of protein for on-chip hydrolysis. Besides generating several internal peptide fragments, limited acid hydrolysis also has the advantage of generating peptide ladders from the N- or C-terminus of the protein. From these ladders, partial primary sequence of the protein can be directly derived when analyzed by a simple laser desorption/ionization mass spectrometer. Furthermore, tandem mass spectrometry can be performed on several internal peptide fragments, thus facilitating the identification of several proteins within a mixture. Based upon the preliminary results of this work, we continue to explore the possibility of using limited acid hydrolysis to identify unknown proteins captured on ProteinChip array surfaces.     

Expression, purification and characterization of the second Kunitz-type protease inhibitor domain of the human WFIKKN protein.

Recently we have described a novel secreted protein (the WFIKKN protein) that consists of multiple types of protease inhibitory modules, including two tandem Kunitz-type protease inhibitor-domains. On the basis of its homologies we have suggested that the WFIKKN protein is a multivalent protease inhibitor that may control the action of different proteases. In the present work we have expressed the second Kunitz-type protease inhibitor domain of the human protein WFIKKN in Escherichia coli, purified it by affinity chromatography on trypsin-Sepharose and its structure was characterized by CD spectroscopy. The recombinant protein was found to inhibit trypsin (Ki = 9.6 nm), but chymotrypsin, elastase, plasmin, pancreatic kallikrein, lung tryptase, plasma kallikrein, thrombin, urokinase or tissue plasminogen activator were not inhibited by the recombinant protein even at 1 microm concentration. In view of the marked trypsin-specificity of the inhibitor it is suggested that its physiological target may be trypsin.     

Automated comparative proteomics based on multiplex tandem mass spectrometry and stable isotope labeling

Comparative proteomic approaches using isotopic labeling and MS have become increasingly popular. Conventionally quantification is based on MS or extracted ion chromatogram (XIC) signals of differentially labeled peptides. However, in these MS-based experiments, the accuracy and dynamic range of quantification are limited by the high noise levels of MS/XIC data. Here we report a quantitative strategy based on multiplex (derived from multiple precursor ions) MS/MS data. One set of proteins was metabolically labeled with [13C6]lysine and [15N4]arginine; the other set was unlabeled. For peptide analysis after tryptic digestion of the labeled proteins, a wide precursor window was used to include both the light and heavy versions of each peptide for fragmentation. The multiplex MS/MS data were used for both protein identification and quantification. The use of the wide precursor window increased sensitivity, and the y ion pairs in the multiplex MS/MS spectra from peptides containing labeled and unlabeled lysine or arginine offered more information for, and thus the potential for improving, protein identification. Protein ratios were obtained by comparing intensities of y ions derived from the light and heavy peptides. Our results indicated that this method offers several advantages over the conventional XIC-based approach, including increased sensitivity for protein identification and more accurate quantification with more than a 10-fold increase in dynamic range. In addition, the quantification calculation process was fast, fully automated, and independent of instrument and data type. This method was further validated by quantitative analysis of signaling proteins in the EphB2 pathway in NG108 cells.     

Identification and quantification of DNA repair proteins by liquid chromatography/isotope-dilution tandem mass spectrometry using their fully 15N-labeled analogues as internal standards

Oxidatively induced DNA damage is implicated in disease, unless it is repaired by DNA repair. Defects in DNA repair capacity may be a risk factor for various disease processes. Thus, DNA repair proteins may be used as early detection and therapeutic biomarkers in cancer and other diseases. For this purpose, the measurement of the expression level of these proteins in vivo will be necessary. We applied liquid chromatography/isotope-dilution tandem mass spectrometry (LC-MS/MS) for the identification and quantification of DNA repair proteins human 8-hydroxyguanine-DNA glycosylase (hOGG1) and Escherichia coli formamidopyrimidine DNA glycosylase (Fpg), which are involved in base-excision repair of oxidatively induced DNA damage. We overproduced and purified (15)N-labeled analogues of these proteins to be used as suitable internal standards to ensure the accuracy of quantification. Unlabeled and (15)N-labeled proteins were digested with trypsin and analyzed by LC-MS/MS. Numerous tryptic peptides of both proteins were identified on the basis of their full-scan mass spectra. These peptides matched the theoretical peptide fragments expected from trypsin digestion and provided statistically significant protein scores that would unequivocally identify these proteins. We also recorded the product ion spectra of the tryptic peptides and defined the characteristic product ions. Mixtures of the analyte proteins and their (15)N-labeled analogues were analyzed by selected-reaction monitoring on the basis of product ions. The results obtained suggest that the methodology developed would be highly suitable for the positive identification and accurate quantification of DNA repair proteins in vivo as potential biomarkers for cancer and other diseases.     

Three recombinant serine proteinase inhibitors expressed from the coding region of the thrombin inhibitor dipetalogastin

Dipetalogastin is a potent thrombin inhibitor from Dipetalogaster maximus. The cDNA of dipetalogastin codes for a large protein which consists of six Kazal-type domains. There are three tandem, homologous regions each including two domains. Three biologically active recombinant proteins rDI, rDII and rDIII each corresponding to one region of the dipetalogastin cDNA were expressed, purified and investigated with regard to their biological activities. rDI and rDII with molecular masses of 12,660 and 12,911 Da, respectively, proved to be potent thrombin inhibitors. The investigation of their influences on amidolytic activities of different serine proteases showed no inhibition of factor Xa (FXa) and alpha-chymotrypsin. At a large molar excess of rDI and rDII over the enzymes only low effects on the activities of trypsin and plasmin were observed. rDIII differs much from the both others. An inhibition of thrombin was found only at a molar excess of rDIII over the enzyme. Furthermore, an inhibition of trypsin and low effects on plasmin were detected at a molar excess of inhibitor over these enzymes. These results indicate that rDIII is active against thrombin, trypsin and plasmin, and finally possesses no specificity for only one serine proteinase.     

Pre‐fractionation of rat liver cytosol proteins prior to mass spectrometry‐based proteomic analysis using tandem biomimetic affinity chromatography

Efficient and high resolution separation of the protein mixture prior to trypsin digestion and mass spectrometry (MS) analysis is generally used to reduce the complexity of samples, an approach that highly increases the probability of detecting low‐copy‐number proteins. Our laboratory has constructed an affinity ligand library composed of thousands of ligands with different protein absorbance effects. Structural differences between these ligands result in different non‐bonded protein–ligand interactions, thus each ligand exhibits a specific affinity to some protein groups. In this work, we first selected out several synthetic affinity ligands showing large band distribution differences in proteins absorbance profiles, and a tandem composition of these affinity ligands was used to distribute complex rat liver cytosol into simple subgroups. Ultimately, all the fractions collected from tandem affinity pre‐fractionation were digested and then analyzed by LC‐MS/MS, which resulted in high confidence identification of 665 unique rat protein groups, 1.8 times as many proteins as were detected in the un‐fractionated sample (371 protein groups). Of these, 375 new proteins were identified in tandem fractions, and most of the proteins identified in un‐fractionated sample (290, 80%) also emerged in tandem fractions. Most importantly, 430 unique proteins (64.7%) only characterized in specific fractions, indicating that the crude tissue extract was well distributed by tandem affinity fractionation. All detected proteins were bioinformatically annotated according to their physicochemical characteristics (such as MW, pI, GRAVY value, TM Helices). This approach highlighted the sensitivity of this method to a wide variety of protein classes. Combined usage of tandem affinity pre‐fractionation with MS‐based proteomic analysis is simple, low‐cost, and effective, providing the prospect of broad application in proteomics. Copyright © 2009 John Wiley & Sons, Ltd.