Design and use of multi-affinity surfaces in biomolecular interaction analysis-mass spectrometry (BIA/MS): a step toward the design of SPR/MS arrays

The feasibility of multi-affinity ligand surfaces in biomolecular interaction analysis-mass spectrometry (BIA/MS) was explored in this work. Multi-protein affinity surfaces were constructed by utilizing antibodies to beta-2-microglobulin, cystatin C, retinol binding protein, transthyretin, serum amyloid P and C-reactive protein. In the initial experiments, all six antibodies were immobilized on a single site (flow cell) on the sensor chip surface, followed by verification of the surface activity via separate injections of purified proteins. After an injection of diluted human plasma aliquot over the antibodies-derivatized surfaces, and subsequent MALDI-TOF MS analysis, signals representing five out of the six targeted proteins were observed in the mass spectra. Further, to avoid the complexity of the spectra, the six proteins were divided into two groups (according to their molecular weight) and immobilized on two separate surfaces on a single sensor chip, followed by an injection of human plasma aliquot. The resulting mass spectra showed signals from all proteins. Also, the convolution resulting from the multiply charged ion species was eliminated. The ability to create such multi-affinity surfaces indicates that smaller-size ligand areas/spots can be employed in the BIA/MS protein interaction screening experiments, and opens up the possibilities for construction of novel multi-arrayed SPR-MS platforms and methods for high-throughput parallel protein interaction investigations.     

Delineating protein-protein interactions via biomolecular interaction analysis-mass spectrometry

The utility of biomolecular interaction analysis-mass spectrometry (BIA/MS) in screening for protein-protein interactions was explored in this work. Experiments were performed in which proteins served as ligands for screening of possible interactions with other proteins from human plasma and urine. The proteins utilized were beta-2-microglobulin, cystatin C (cysC), retinol binding protein (RBP), transthyretin (TTR), alpha-1-microglobulin, C-reactive protein, transferrin and papain. The immobilization of functionally active proteins was confirmed via interactions with antibodies to the corresponding proteins. Various dilutions of human urine and plasma were injected over the protein-derivatized surfaces. It was observed that the urine injections generally yielded smaller SPR responses than those observed after the plasma injections. The BIA/MS experiments did not reveal novel protein-protein interactions, although several established interactions (such as those between RBP and TTR, and cysC and papain) were validated. Few protein ligand deficiencies (such as truncations) leading to false negative and false positive BIA/MS results were also discovered.     

Immuno‐MALDI‐TOF MS: New perspectives for clinical applications of mass spectrometry

The discovery of novel biomarkers by means of advanced detection tools based on proteomic analysis technologies necessitates the development of improved diagnostic methods for application in clinical routine. On the basis of three different application examples, this review presents the limitations of conventional routine diagnostic assays and illustrates the advantages of immunoaffinity enrichment combined with MALDI‐TOF MS. Applying this approach increases the specificity of the analysis supporting a better diagnostic recognition, sensitivity, and differentiation of certain diseases. The use of MALDI‐TOF MS as detection method facilitates the identification of modified peptides and proteins providing additional information. Further, employing respective internal standard peptides allows for relative and absolute quantitation which is mandatory in the clinical context. Although MALDI‐TOF MS is not yet established for clinical routine diagnostics this technology has a high potential for improvement of clinical diagnostics and monitoring therapeutic efficacy.     

Analysis of native proteins from biological fluids by biomolecular interaction analysis mass spectrometry (BIA/MS): exploring the limit of detection, identification of non-specific binding and detection of multi-protein complexes

Biomolecular interaction analysis mass spectrometry (BIA/MS) is a two-dimensional analytical technique that quantitatively and qualitatively detects analytes of interests. In the first dimension, surface plasmon resonance (SPR) is utilized for detection of biomolecules in their native environment. Because SPR detection is non-destructive, analyte(s) retained on the SPR-active sensor surface can be analyzed in a second dimension using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The qualitative nature of the MALDI-TOF MS analysis complements the quantitative character of SPR sensing and overcomes the shortcomings of the SPR detection stemming from the inability to differentiate and characterize multi-protein complexes and non-specific binding. In this work, the benefit of performing MS analysis following SPR sensing is established. Retrieval and detection of four markers present in biological fluids (cystatin C, beta-2-microglobulin, urinary protein 1 and retinol binding protein) was explored to demonstrate the effectiveness of BIA/MS in simultaneous detection of clinically related biomarkers and delineation of non-specific binding. Furthermore, the BIA/MS limit of detection at very low SPR responses was investigated. Finally, detection of in-vivo assembled protein complexes was achieved for the first time using BIA/MS.     

Labeling elastase digests with TMT: informational gain by identification of poorly detectable peptides with MALDI-TOF/TOF mass spectrometry

The applicability of the less specific protease elastase for the identification of membrane and cytosolic proteins has already been demonstrated. MALDI as ionization technique particularly favors the detection of basic and to a lesser extent of weakly acidic peptides, whereas neutral peptides often remain undetected. Moreover, peptides below 700 Da are routinely excluded. In the following study, the advantage of additional information gained from tandem mass tag zero labeled peptides and the resultant increase in sequence coverage was evaluated. Through derivatization with tandem mass tag reagents, peptide measurement within the standard mass range of the MALDI reflector mode is achievable due to the mass increase. Compared to the unlabeled sample, peptides exhibiting relatively low molecular masses, pI values or higher hydrophobicity could be identified.     

Surface plasmon resonance mass spectrometry in proteomics

Due to the enormous complexity of the proteome, focus in proteomics shifts more and more from the study of the complete proteome to the targeted analysis of part of the proteome. The isolation of this specific part of the proteome generally includes an affinity-based enrichment. Surface plasmon resonance (SPR), a label-free technique able to follow enrichment in real-time and in a semiquantitative manner, is an emerging tool for targeted affinity enrichment. Furthermore, in combination with mass spectrometry (MS), SPR can be used to both selectively enrich for and identify proteins from a complex sample. Here we illustrate the use of SPR-MS to solve proteomics-based research questions, describing applications that use very different types of immobilized components: such as small (drug or messenger) molecules, peptides, DNA and proteins. We evaluate the current possibilities and limitations and discuss the future developments of the SPR-MS technique.     

Protein prenylation in an insect cell-free protein synthesis system and identification of products by mass spectrometry

To evaluate the ability of an insect cell-free protein synthesis system to carry out proper protein prenylation, several CAIX (X indicates any C-terminal amino acid) sequences were introduced into the C-terminus of truncated human gelsolin (tGelsolin). Tryptic digests of these mutant proteins were analyzed by MALDI-TOF MS and MALDI-quadrupole-IT-TOF MS. The results indicated that the insect cell-free protein synthesis system possesses both farnesyltransferase (FTase) and geranylgeranyltransferase (GGTase) I, as is the case of the rabbit reticulocyte lysate system. The C-terminal amino acid sequence requirements for protein prenylation in this system showed high similarity to those observed in rat prenyltransferases. In the case of rhoC, which is a natural geranylgeranylated protein, it was found that it could serve as a substrate for both prenyltransferases in the presence of either farnesyl or geranylgeranyl pyrophosphate, whereas geranylgeranylation was only observed when both prenyl pyrophosphates were added to the in vitro translation reaction mixture. Thus, a combination of the cell-free protein synthesis system with MS is an effective strategy to analyze protein prenylation.     

Annexin A2/P11 interaction: new insights into annexin A2 tetramer structure by chemical crosslinking, high-resolution mass spectrometry, and computational modeling

During the past few years, the structural analysis of proteins and protein complexes by chemical crosslinking and mass spectrometry has enjoyed increasing popularity. With this approach we have investigated the quaternary structure of the complex between annexin A2 and p11, which is involved in numerous cellular processes. Although high-resolution data are available for both interaction partners as well as for the complex between two p11 subunits and two annexin A2 N-terminal peptides, the structure of the complete annexin A2/p11 heterotetramer has not yet been solved at high resolution. Thus, the quaternary structure of the biologically relevant, membrane-bound annexin A2/p11 complex is still under discussion, while the existence of a heterotetramer or a heterooctamer is the prevailing opinion. We gained further insight into the spatial organization of the annexin A2/p11 heterotetramer by employing chemical crosslinking combined with high-resolution mass spectrometry. Furthermore, tandem mass spectrometry served as a tool for an exact localization of crosslinked amino acid residues and for a confirmation of crosslinked product assignment. On the basis of distance constraints from the crosslinking data we derived structural models of the annexin A2/p11 heterotetramer by computational docking with Rosetta. We propose an octameric model for the annexin A2/p11 complex, which exerts annexin A2 function. The proposed structure of the annexin A2/p11 octamer differs from so far suggested models and sheds new light into annexin A2/p11 interaction.     

Evaluation of VITEK matrix‐assisted laser desorption ionization–time of flight mass spectrometry for identification of anaerobes

Rapid and adequate identification of anaerobic bacterial species still presents a challenge for most diagnostic laboratories, hindering the selection of appropriate therapy. In this study, the identification capacity of 16S rRNA sequence analysis, VITEK 2 (BioMérieux, Lyon, France) compact analysis and VITEK MS‐mediated identification for anaerobic bacterial species was compared. Eighty‐five anaerobic bacterial isolates from 11 provinces in China belonging to 14 genera were identified by these three methods. Differences in identification between these three methods were compared. Consistent identification results were obtained for 54 (54/85, 63.5%) isolates by all three methods, the most discordant results being concentrated in Clostridium XI (n = 8) and Bacteroides fragilis (n = 9) clusters. Using the VITEK MS system, 74 (74/90, 82.2%) isolates were identified as single species consistent with 16S rRNA sequence analysis, which was significantly better than the results obtained with VITEK 2 Compact (P < 0.01). Misidentifications by the Vitek 2 Compact and Vitek MS systems were mainly observed in the Clostridium XI (n = 8)and B. fragilis clusters (n = 9). VITEK MS identified anaerobic bacteria even after they had been exposed to oxygen for a week. Identification by the Vitek MS system was more consistent with 16S rRNA sequence analysis than identification by Vitek 2 Compact. Continuous expansion of the VITEK MS database with rare described anaerobic species is warranted to improve both the efficiency and accuracy of VITEK MS identification in routine diagnostic microbiology.     

N-Terminal protein modifications in an insect cell-free protein synthesis system and their identification by mass spectrometry

To evaluate the ability of an insect cell-free protein synthesis system to generate proper N-terminal cotranslational protein modifications such as removal of the initiating Met, N-acetylation, and N-myristoylation, several mutants were constructed using truncated human gelsolin (tGelsolin) as a model protein. Tryptic digests of these mutants were analyzed by MALDI-TOF MS and MALDI-quadrupole-IT-TOF MS. The wild-type tGelsolin, which is an N-myristoylated protein, was found to be N-myristoylated when myristoyl-CoA was added to the in vitro translation reaction mixture. N-myristoylation did not occur on the Gly-2 to Ala mutant, in which the N-myristoylation motif was disrupted, whereas this mutant was found to be N-acetylated after removal of the initiating Met. Analyses of Gly-2 to His and Leu-3 to Asp mutants revealed that the amino acids at positions 2 and 3 strongly affect the susceptibility of the nascent peptide chain to removal of the initiating Met and to N-acetylation, respectively. These results suggest that N-terminal modifications occurring in the insect cell-free protein synthesis system are quite similar to those observed in the mammalian protein synthesis system. Thus, a combination of the cell-free protein synthesis system with MS is an effective strategy to analyze protein modifications.     

Characterization of antibody-antigen interactions: comparison between surface plasmon resonance measurements and high-mass matrix-assisted laser desorption/ionization mass spectrometry

The interaction between the bovine prion protein (bPrP) and a monoclonal antibody, 1E5, was studied with high-mass matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) and surface plasmon resonance (SPR). In the case of MS a cross-linking stabilization was used prior to the analysis, whereas for SPR the antibody was immobilized and bPrP was injected. We compared the determination of parameters such as the epitope, the kinetics and binding strength, and the capacity of the antigen to bind two different antibodies. The two methods are highly complementary. SPR measurements require a lower amount of sample but are more time-consuming due to all of the necessary side steps (e.g., immobilization, regeneration). High-mass MALDI MS needs a higher overall amount of sample and cannot give direct access to the kinetic constants, but the analysis is faster and easier compared with SPR.     

PAS‐cal: A repetitive peptide sequence calibration standard for MALDI mass spectrometry

Mass spectrometers equipped with matrix‐assisted laser desorption/ionization (MALDI‐MS) require frequent multipoint calibration to obtain good mass accuracy over a wide mass range and across large numbers of samples. In this study, we introduce a new synthetic peptide mass calibration standard termed PAS‐cal tailored for MALDI‐MS based bottom‐up proteomics. This standard consists of 30 peptides between 8 and 37 amino acids long and each constructed to contain repetitive sequences of Pro, Ala and Ser as well as one C‐terminal arginine residue. MALDI spectra thus cover a mass range between 750 and 3200 m/z in MS mode and between 100 and 3200 m/z in MS/MS mode. Our results show that multipoint calibration of MS spectra using PAS‐cal peptides compares well to current commercial reagents for protein identification by PMF. Calibration of tandem mass spectra from LC‐MALDI experiments using the longest peptide, PAS‐cal37, resulted in smaller fragment ion mass errors, more matching fragment ions and more protein and peptide identifications compared to commercial standards, making the PAS‐cal standard generically useful for bottom‐up proteomics.     

Extending ribosomal protein identifications to unsequenced bacterial strains using matrix-assisted laser desorption/ionization mass spectrometry

A protocol has been developed that allows protein identifications using available DNA-based or protein sequences from a reference strain of a bacterial species to be extended to bacterial strains for which no prior DNA-based or protein sequence information exists. The protocol is predicated on careful isolation of a specific sub-cellular group of proteins. In this study, ribosomal proteins were chosen due to their high relative abundance and similarity in copy number per cell. After isolation of ribosomal proteins, MALDI-MS is used to acquire accurate protein molecular weights. An iterative comparison of reference protein molecular weights and identities is made to the resulting data, allowing for the straightforward identification of ribosomal proteins from any non-reference strains. This approach can reveal differences between proteins at the amino acid or post-translational level. The protocol was developed, validated and applied to ribosomal proteins from three strains of the extreme thermophile Thermus thermophilus. This approach revealed that nearly 60% of the ribosomal proteins from all three strains are identical. The extension of protein identification to additional bacterial strains can be useful in phylogenetic studies as well as in biomarker identification.     

Proteomic analysis of cold adaptation in a Siberian permafrost bacterium--Exiguobacterium sibiricum 255-15 by two-dimensional liquid separation coupled with mass spectrometry

Bacterial cold adaptation in Exiguobacterium sibiricum 255-15 was studied on a proteomic scale using a 2-D liquid phase separation coupled with MS technology. Whole-cell lysates of E. sibiricum 255-15 grown at 4 degrees C and 25 degrees C were first fractionated according to pI by chromatofocusing (CF), and further separated based on hydrophobicity by nonporous silica RP HPLC (NPS-RP-HPLC) which was on-line coupled with an ESI-TOF MS for intact protein M(r) measurement and quantitative interlysate comparison. Mass maps were created to visualize the differences in protein expression between different growth temperatures. The differentially expressed proteins were then identified by PMF using a MALDI-TOF MS and peptide sequencing by MS/MS with a MALDI quadrupole IT TOF mass spectrometer (MALDI-QIT-TOF MS). A total of over 500 proteins were detected in this study, of which 256 were identified. Among these proteins 39 were cold acclimation proteins (Caps) that were preferentially or uniquely expressed at 4 degrees C and three were homologous cold shock proteins (Csps). The homologous Csps were found to be similarly expressed at 4 degrees C and 25 degrees C, where these three homologous Csps represent about 10% of the total soluble proteins at both 4 degrees C and 25 degrees C.     

Selective detection of 2-nitrobenzenesulfenyl-labeled peptides by matrix-assisted laser desorption/ionization-time of flight mass spectrometry using a novel matrix

The 2-nitrobenzenesulfenyl (NBS) method, which is useful for quantitative proteome analysis, is based on stable isotope labeling of tryptophan residues with NBS chloride ((12)C(6)-NBSCl or (13)C(6)-NBSCl). We found that 3-hydroxy-4-nitrobenzoic acid (3H4NBA) is a more suitable matrix than 2,5-dihydroxybenzoic acid (DHB) for detecting NBS-labeled peptides by MALDI-quadrupole IT (QIT)-TOF MS . Furthermore, NBS-labeled peptides were selectively ionized and detected in a mixture of NBS-labeled and unlabeled peptides. Labeled paired peaks were easily detected without enrichment, nonpaired labeled peaks were clearly distinguished from unlabeled contaminating peptides, and nitrotyrosine-containing peptides were also selectively detected on the 3H4NBA matrix, while by-product-peaks arising from nitrobenzene moieties were suppressed. The use of 3H4NBA as a comatrix with CHCA improved the sensitivity of detection while substantially retaining the selectivity of 3H4NBA. The 3H4NBA matrix offers great advantages in terms of simplicity, sensitivity, and usability when used for the NBS method and for MALDI-TOF MS analysis applied to compounds having a nitrobenzene ring.     

Identification of novel p53-binding proteins by biomolecular interaction analysis combined with tandem mass spectrometry

Electrospray tandem mass spectrometry (ESI-MS/MS) was combined with biomolecular interaction analysis (BIA) to develop a method of direct protein identification after real-time analysis of protein-protein interactions. Using this method, called BIA-MS/MS, we detected multiple p53-interacting proteins in whole tissue extracts from human placenta and liver. Peptide sequencing revealed three proteins whose interaction with p53 had not been previously reported: a cyclin-dependent kinase inhibitor p57/Kip2, a serine/threonine protein phosphatase PP1C, and hemoglobin. Using our system, unambiguous sequence information can be obtained at the femto- to picomole level after repeating the recovery procedure five times. Furthermore, the association and dissociation constants are easily determined by kinetic analysis. This system provides a powerful tool for analyzing complex biological materials in a simple but highly specific and sensitive manner.     

Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry as a useful tool for the rapid identification of wild flea vectors preserved in alcohol

Flea identification is a significant issue because some species are considered as important vectors of several human pathogens that have emerged or re‐emerged recently, such as Bartonella henselae (Rhizobiales: Bartonellaceae) and Rickettsia felis (Rickettsiales: Rickettsiaceae). Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) has been evaluated in recent years for the identification of multicellular organisms, including arthropods. A preliminary study corroborated the usefulness of this technique for the rapid identification of fleas, creating a preliminary database containing the spectra of five species of flea. However, longterm flea preservation in ethanol did not appear to be an adequate method of storage in the context of specimen identification by MALDI‐TOF MS profiling. The goal of the present work was to assess the performance of MALDI‐TOF MS in the identification of seven flea species [Ctenocephalides felis (Siphonaptera: Pulicidae), Ctenocephalides canis, Pulex irritans (Siphonaptera: Pulicidae), Archaeopsylla erinacei (Siphonaptera: Pulicidae), Leptopsylla taschenbergi (Siphonaptera: Ceratophyllidae), Stenoponia tripectinata (Siphonaptera: Stenoponiidae) and Nosopsyllus fasciatus (Siphonaptera: Ceratophyllidae)] collected in the field and stored in ethanol for different periods of time. The results confirmed that MALDI‐TOF MS can be used for the identification of wild fleas stored in ethanol. Furthermore, this technique was able to discriminate not only different flea genera, but also the two congeneric species C. felis and C. canis.     

Integration of surface plasmon resonance with mass spectrometry: automated ligand fishing and sample preparation for MALDI MS using a Biacore 3000 biosensor.

Integrating surface plasmon resonance analysis with mass spectrometry allows detection and characterization of molecular interactions to be complemented with identification of interaction partners. We have developed a procedure for Biacore 3000 that automatically performs all steps from ligand fishing and recovery to sample preparation for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry including on-target digestion. In the model system used in this study a signal transduction protein, calmodulin, was selectively captured from brain extract by one of its interaction partners immobilized on a sensor chip. The bound material was eluted, deposited directly onto a MALDI target, and analyzed by mass spectrometry both as an intact protein and after on-target tryptic digestion. The procedure with direct deposition of recovered material on the MALDI target reduces sample losses and, in combination with automatic sample processing, increases the throughput of surface plasmon resonance mass spectrometry analysis.