Rapid typing of bacteria using matrix-assisted laser desorption ionisation time-of-flight mass spectrometry and pattern recognition software.

Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) of intact microorganisms, also known as intact cell MALDI-TOF-MS (ICM-MS), has been shown to produce characteristic mass spectral fingerprints of moieties desorbed from the cell surface. ICM-MS spectra can be obtained in minutes after removal of a colony from a culture plate. The similarity of ICM-MS spectra of replicate samples and of two different batches of the same bacterial strain demonstrates, in this study, the reproducibility of the technique. We have developed the Manchester Metropolitan University Search Engine (MUSE) to rapidly build and search databases of ICM-MS spectra. A database of 35 strains, representing 20 species and 12 genera, was built with MUSE and used to identify 212 isolates. The database was created in 26 s and loaded in 10 s, ready for searching, which took less than 1 s per isolate. Correct matches were made in 79%, 84% and 89% of the 212 samples at strain, species and genus levels, respectively. At least 50% of the replicates of 42 of the 45 isolates matched the correct strain, and the most commonly identified species for 43 of the 45 isolates was the correct one. The close match of the Escherichia coli strains containing the O157 antigen and the E. coli strains containing the K1 antigen suggests that these antigens may have a dominating influence on the ICM-MS fingerprints of these strains. We now have the ability to acquire ICM-MS fingerprints of bacteria and to search a database of these fingerprints within minutes, so that the rapid identification of bacteria to the strain level can be realised.     

Direct identification of ubiquitination sites on ubiquitin-conjugated CHIP using MALDI mass spectrometry

The study of protein ubiquitination, a post-translational modification by ubiquitin, has emerged as one of the most active areas in biology because of the important role of this type of modification on the regulation of various cellular proteins. Advances in techniques for the determination and site mapping of protein ubiquitination can facilitate the elucidation of molecular mechanisms of this modification. We have recently described a novel method for identifying peptides containing ubiquitinated amino acid residues, based on the MALDI-MS/MS analysis of tryptic peptide derivatives. In particular, we have utilized N-terminal sulfonation of these peptides to provide a unique fragmentation pattern that leads to the direct identification and sequencing of ubiquitin modified peptides. Here we present an application of this new method on the characterization of ubiquitin conjugated C-terminal Hsc70-interacting protein (CHIP), a recently identified U-box containing E3 enzyme. Three peptides bearing ubiquitination sites have been identified from the digest of ubiquitinated CHIP; one of these was a site on CHIP, while the other two were found on the ubiquitin molecules, demonstrating that sulfonation of tryptic peptides is a general and efficient method for characterizing protein ubiquitination.     

Large-scale protein identification using mass spectrometry

Recent achievements in genomics have created an infrastructure of biological information. The enormous success of genomics promptly induced a subsequent explosion in proteomics technology, the emerging science for systematic study of proteins in complexes, organelles, and cells. Proteomics is developing powerful technologies to identify proteins, to map proteomes in cells, to quantify the differential expression of proteins under different states, and to study aspects of protein-protein interaction. The dynamic nature of protein expression, protein interactions, and protein modifications requires measurement as a function of time and cellular state. These types of studies require many measurements and thus high throughput protein identification is essential. This review will discuss aspects of mass spectrometry with emphasis on methods and applications for large-scale protein identification, a fundamental tool for proteomics.     

Rapid identification of ubiquitination and SUMOylation target sites by microfluidic peptide array

SUMOylation and ubiquitination are two essential post translational modifications (PTMs) involved in the regulation of important biological processes in eukaryotic cells. Identification of ubiquitin (Ub) and small ubiquitin-related modifier (SUMO)-conjugated lysine residues in proteins is critical for understanding the role of ubiquitination and SUMOylation, but remains experimentally challenging. We have developed a powerful in vitro Ub/SUMO assay using a novel high density peptide array incorporated within a microfluidic device that allows rapid identification of ubiquitination and SUMOylation sites on target proteins. We performed the assay with a panel of human proteins and a microbial effector with known target sites for Ub or SUMO modifications, and determined that 80% of these proteins were modified by Ub or specific SUMO isoforms at the sites previously determined using conventional methods. Our results confirm the specificity for both SUMO isoform and individual target proteins at the peptide level. In summary, this microfluidic high density peptide array approach is a rapid screening assay to determine sites of Ub and SUMO modification of target substrates, which will provide new insights into the composition, selectivity and specificity of these PTM target sites.     

Classification and identification of toxic polypeptides using a pattern recognition method

A crucial rule for both rapid identification and classification of toxins by the relative content of the five most informative amino acids (glycine, cysteine, threonine, alanine and tryptophane) was established by using a computer algorithm of pattern recognition based on amino acid sequences of three families of toxic polypeptides.     

Identification of precise electrostatic recognition sites between cytochrome c6 and the photosystem I subunit PsaF using mass spectrometry

The reduction of the photo-oxidized special chlorophyll pair P700 of photosystem I (PSI) in the photosynthetic electron transport chain of eukaryotic organisms is facilitated by the soluble copper-containing protein plastocyanin (pc). In the absence of copper, pc is functionally replaced by the heme-containing protein cytochrome c6 (cyt c6) in the green alga Chlamydomonas reinhardtii. Binding and electron transfer between both donors and PSI follows a two-step mechanism that depends on electrostatic and hydrophobic recognition between the partners. Although the electrostatic and hydrophobic recognition sites on pc and PSI are well known, the precise electrostatic recognition site on cyt c6 is unknown. To specify the interaction sites on a molecular level, we cross-linked cyt c6 and PSI using a zero-length cross-linker and obtained a cross-linked complex competent in fast and efficient electron transfer. As shown previously, cyt c6 cross-links specifically with the PsaF subunit of PSI. Mass spectrometric analysis of tryptic peptides from the cross-linked product revealed specific interaction sites between residues Lys27 of PsaF and Glu69 of cyt c6 and between Lys23 of PsaF and Glu69/Glu70 of cyt c6. Using these new data, we present a molecular model of the intermolecular electron transfer complex between eukaryotic cyt c6 and PSI.     

Characterization of purified c-type heme-containing peptides and identification of c-type heme-attachment sites in Shewanella oneidenis cytochromes using mass spectrometry

We describe methods for mass spectrometric identification of heme-containing peptides from c-type cytochromes that contain the CXXCH (X=any amino acid) sequence motif. The heme fragment ion yielded the most abundant MS/MS peak for standard heme-containing peptides with one amino acid difference for both 2+ and 3+ peptide charge states; both sequence and charge affect the extent of heme loss. Application to Shewanella oneidenis demonstrated the utility of this approach for identifying c-type heme-containing peptides from complex proteome samples.     

Rapid identification of fluorochrome modification sites in proteins by LC ESI-Q-TOF mass spectrometry

Conjugation of either a fluorescent dye or a drug molecule to the ε-amino groups of lysine residues of proteins has many applications in biology and medicine. However, this type of conjugation produces a heterogeneous population of protein conjugates. Because conjugation of fluorochrome or drug molecule to a protein may have deleterious effects on protein function, the identification of conjugation sites is necessary. Unfortunately, the identification process can be time-consuming and laborious; therefore, there is a need to develop a rapid and reliable way to determine the conjugation sites of the fluorescent label or drug molecule. In this study, the sites of conjugation of fluorescein-5'-isothiocyanate and rhodamine-B-isothiocyanate to free amino groups on the insert-domain (I-domain) protein derived from the α-subunit of lymphocyte function-associated antigen-1 (LFA-1) were determined by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF MS) along with peptide mapping using trypsin digestion. A reporter fragment of the fluorochrome moiety that is generated in the collision cell of the Q-TOF without explicit MS/MS precursor selection was used to identify the conjugation site. Selected ion plots of the reporter ion readily mark modified peptides in chromatograms of the complex digest. Interrogation of theses spectra reveals a neutral loss/precursor pair that identifies the modified peptide. The results show that one to seven fluorescein molecules or one to four rhodamine molecules were attached to the lysine residue(s) of the I-domain protein. No modifications were found in the metal ion-dependent adhesion site (MIDAS), which is an important binding region of the I-domain.     

Characterization of the glycosylation sites in cyclooxygenase-2 using mass spectrometry

Cyclooxygenase is involved in the biosynthesis and function of prostaglandins. It is a glycoprotein located in the endoplasmic reticulum and in the nuclear envelope, and it has been found to have two isoforms termed COX-1 and COX-2. This paper reports on the glycosylation site analysis of recombinant COX-2 using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) and nanoelectrospray (nanoESI) quadrupole-TOF (Q-TOF) MS. The nanoESI MS analysis of COX-2 revealed the presence of three glycoforms at average molecular masses of 71.4, 72.7, and 73.9 kDa. Each glycoform contained a number of peaks differing by 162 Da indicating heterogeneity and suggesting the presence of high-mannose sugars. The masses of the glycoforms indicate that oligosaccharides occupy two to four sites and a single N-acetylglucosamine (GlcNAc) residue occupied up to two sites. The MALDI MS analysis of a tryptic digest of the protein showed a number of potential glycopeptides. The peptides differed by 162 Da which further suggested high-mannose sugars. Nanoelectrospray MS/MS experiments confirmed glycosylation at the Asn 53 and Asn 130 sites and confirmed the presence of the peptides Asn 396-Arg 414 + GlcNAc and Thr 576-Arg 587 + GlcNAc containing Asn 580. It was not possible to conclusively determine whether the Asn 396 site was glycosylated via an MS/MS experiment, so the tryptic digest was deglycosylated to confirm the presence of the glycopeptides. Finally, a non-glycosylated tryptic peptide was observed containing the Asn 592.     

Automated identification of field-recorded songs of four British grasshoppers using bioacoustic signal recognition

Recognition of Orthoptera species by means of their song is widely used in field work but requires expertise. It is now possible to develop computer-based systems to achieve the same task with a number of advantages including continuous long term unattended operation and automatic species logging. The system described here achieves automated discrimination between different species by utilizing a novel time domain signal coding technique and an artificial neural network. The system has previously been shown to recognize 25 species of British Orthoptera with 99% accuracy for good quality sounds. This paper tests the system on field recordings of four species of grasshopper in northern England in 2002 and shows that it is capable of not only correctly recognizing the target species under a range of acoustic conditions but also of recognizing other sounds such as birds and man-made sounds. Recognition accuracies for the four species of typically 70-100% are obtained for field recordings with varying sound intensities and background signals.     

Rapid protein identification using direct infusion nanoelectrospray ionization mass spectrometry

Current protein identification techniques are largely based on MALDI-TOF mass fingerprinting and LC-ESI MS/MS sequence tag analysis. Here we describe an improved method for rapid protein identification that uses direct infusion nanoelectrospray quadrupole time-of-flight (nanoESI QTOF) MS. Protein digests were analyzed without LC separation using nanoESI on a QSTAR XL MS/MS system in information dependent data acquisition mode. The protein identification conditions and parameters were extensively evaluated with in-solution and in-gel digested protein samples. Rapid identification of proteins was achieved and compared directly to the results obtained on the same samples using nanoflow HPLC-MS/MS on the QSTAR system. The increased throughput, reproducibility, the high data quality, and the ease of use make the direct infusion system an efficient and affordable technique for protein identification analysis.     

Ozone-induced damage of fibrinogen molecules: identification of oxidation sites by high-resolution mass spectrometry

Fibrinogen is highly susceptible to oxidation compared to other plasma proteins. Fibrinogen oxidation damages its structure and affects the protein function. Ozone-induced oxidative modifications of the fibrinogen Aα, Bβ, and γ polypeptide chains upon addition of various amounts of the oxidiser were studied by mass spectrometry. Amino acid residues located on all three chains and main structural parts of the protein were revealed to be involved in oxidation. The αC-connector was shown to be most vulnerable to oxidation as compared to other structural parts while the E region turned out to be the most protected area of the protein. For the first time, it was established that numerous amino acid residues responsible for the conversion of fibrinogen to fibrin remain unaffected upon fibrinogen oxidation. The data obtained in this study indicate that none of the identified residues, which are considered crucial for the binding of both hole “a” and hole “b” to knob “A” and knob “B”, respectively, as well as those responsible for the thrombin binding to fibrinogen E region, have been subjected to chemical alterations under moderate oxidation. The data on fibrinogen oxidation acquired in the current study enable one to assume that some of the structural fibrinogen parts and easily oxidisable residues could be endowed with antioxidant properties. New findings presented here could be essential for the detection of adaptive molecular mechanisms capable of mitigating the detrimental action of reactive oxygen species (ROS) on the functioning of oxidatively damaged fibrinogen. Data are available via ProteomeXchange with identifier PXD012046.

• Highlights

• Various oxidative modifications were detected in fibrinogen by mass spectrometry

• αC-connector has been shown to be most susceptible to oxidation

• E region proved to be least vulnerable to the action of the oxidising agent

• Some of the Met residues in the fibrinogen structure could operate as ROS scavengers

     

Determining heparan sulfate structure in the vicinity of specific sulfotransferase recognition sites by mass spectrometry

Sulfated motifs on heparan sulfate (HS) are involved in various extracellular processes from cell signaling to enzymatic regulation, but the structures of these motifs are obscure. We have developed a strategy to determine the structure of sulfotransferase recognition sites which constitute these motifs. Stable isotope is first introduced into specific sites on HS with HS sulfotransferases and the modified HS is then digested into oligosaccharides of differing sizes. The overlapping oligosaccharides containing the introduced stable isotope are identified by changes in the m/z profiles by mass spectrometry, and their relationships are elucidated. In this way, the HS structures in the vicinity of the sulfotransferase recognition site are quickly determined and groups on precursor structures of HS that direct the action of HS sulfotransferases are pinpointed.     

Rapid identification of acetic acid bacteria using MALDI-TOF mass spectrometry fingerprinting

Acetic acid bacteria (AAB) are widespread microorganisms characterized by their ability to transform alcohols and sugar-alcohols into their corresponding organic acids. The suitability of matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF MS) for the identification of cultured AAB involved in the industrial production of vinegar was evaluated on 64 reference strains from the genera Acetobacter, Gluconacetobacter and Gluconobacter. Analysis of MS spectra obtained from single colonies of these strains confirmed their basic classification based on comparative 16S rRNA gene sequence analysis. MALDI-TOF analyses of isolates from vinegar cross-checked by comparative sequence analysis of 16S rRNA gene fragments allowed AAB to be identified, and it was possible to differentiate them from mixed cultures and non-AAB. The results showed that MALDI-TOF MS analysis was a rapid and reliable method for the clustering and identification of AAB species.     

Automated analysis of mouse serum peptidome using restricted access media and nanoliquid chromatography-tandem mass spectrometry

We demonstrate use of restricted access media with reversed phase functionality (RAM-RP) for analysis of low molecular weight proteins and peptides in mouse serum (75 μl) using a custom designed modular automated processing system (MAPS). RAM-RP fractionation with simultaneous removal of high molecular weight and high abundance proteins is integrated with a follow-on buffer exchange module (BE) to ensure compatibility with subsequent processing steps (trypsin digestion and intact peptide separation prior to mass spectrometric analysis). The high sample capacity afforded by chromatographic methods generates enough sample to achieve comprehensive serum peptidome identification (357 proteins) through tandem mass spectrometric analysis of both intact and digested peptides. Sample losses during transfer between modules are minimized through precise fluidic control; no clogging occurred over several months of serum processing in our low back pressure system. Computer controlled operation of both modules and thorough optimization yield excellent run-to-run reproducibility and protein/peptide overlap in analytical repeats. The robustness of our results demonstrate that the RAM-RP-BE workflow executed on our MAPS platform shows tremendous potential for high throughput peptidome processing, particularly with regard to direct analysis of small-volume serum samples.     

Analysis of lipids from crude lung tissue extracts by desorption electrospray ionization mass spectrometry and pattern recognition

A method is described using desorption electrospray ionization (DESI) mass spectrometry (MS) to obtain phospholipid mass spectral profiles from crude lung tissue extracts. The measured DESI mass spectral lipid fingerprints were then analyzed by unsupervised learning principal components analysis (PCA). This combined approach was used to differentiate the effect(s) of two vaccination routes on lipid composition in mouse lungs. Specifically, the two vaccination routes compared were intranasal (i.n.) and intradermal (i.d.) inoculation of the Francisella tularensis live vaccine strain (Ft–LVS). Lung samples of control and LVS-inoculated mice were quickly extracted with a methanol/chloroform solution, and the crude extract was directly analyzed by DESI–MS, with a total turnaround time of less than 10 min/sample. All of the measured DESI mass spectra (in both positive and negative ion mode) were compared via PCA, resulting in clear differentiation of mass spectral profiles of i.n.-inoculated mouse lung tissues from those of i.d.-inoculated and control mouse lung tissues. Lipid biomarkers responsible for sample differentiation were identified via tandem MS (MS/MS) measurements or by comparison with mass spectra of lipid standards. The DESI–MS approach described here provided a practical and rapid means to analyze tissue samples without extensive extractions and solvent changes.