Multi-Enzymatic Limited Digestion: The Next-Generation Sequencing for Proteomics?

Over the past 40 years, proteomics, generically defined as the field dedicated to the identification and analysis of proteins, has tremendously gained in popularity and potency through advancements in genome sequencing, separative techniques, mass spectrometry, and bioinformatics algorithms. As a consequence, its scope of application has gradually enlarged and diversified to meet specialized topical biomedical subjects. Although the tryptic bottom-up approach is widely regarded as the gold standard for rapid screening of complex samples, its application for precise and confident mapping of protein modifications is often hindered due to partial sequence coverage, poor redundancy in indicative peptides, and lack of method flexibility. We here show how the synergic and time-limited action of a properly diluted mix of multiple enzymes can be exploited in a versatile yet straightforward protocol to alleviate present-day drawbacks. Merging bottom-up and middle-down ideologies, our results highlight broad assemblies of overlapping peptides that enable refined and reliable characterizations of proteins, including variant identification, and their carried modifications, including post-translational modifications, truncations, and cleavages. Beyond this boost in performance, our methodology also offers efficient de novo sequencing capabilities, in view of which we here present a dedicated custom assembly algorithm.

Mass Spectrometry Imaging and Identification of Peptides Associated with Cephalic Ganglia Regeneration in Schmidtea mediterranea.

Tissue regeneration is a complex process that involves a mosaic of molecules that vary spatially and temporally. Insights into the chemical signaling underlying this process can be achieved with a multiplex and untargeted chemical imaging method such as mass spectrometry imaging (MSI), which can enablede novostudies of nervous system regeneration. A combination of MSI and multivariate statistics was used to differentiate peptide dynamics in the freshwater planarian flatwormSchmidtea mediterraneaat different time points during cephalic ganglia regeneration. A protocol was developed to makeS. mediterraneatissues amenable for MSI. MS ion images of planarian tissue sections allow changes in peptides and unknown compounds to be followed as a function of cephalic ganglia regeneration. In conjunction with fluorescence imaging, our results suggest that even though the cephalic ganglia structure is visible after 6 days of regeneration, the original chemical composition of these regenerated structures is regained only after 12 days. Differences were observed in many peptides, such as those derived from secreted peptide 4 and EYE53-1. Peptidomic analysis further identified multiple peptides from various known prohormones, histone proteins, and DNA- and RNA-binding proteins as being associated with the regeneration process. Mass spectrometry data also facilitated the identification of a new prohormone, which we have named secreted peptide prohormone 20 (SPP-20), and is up-regulated during regeneration in planarians.

Quantification of transferrin in human serum using both QconCAT and synthetic internal standards.

Transferrin, an iron transport protein, is a clinically important biomarker in diseases such as iron-deficiency anemia. Current diagnostic methods for transferrin levels lack quantitative accuracy, suggesting the need for alternative approaches like LC-MS with isotope-labeled peptides as internal standards. Besides solid-phase synthesis, isotope-labeled peptides are also generated by a method called QconCAT where peptides are expressed from DNA in the presence of heavy isotope media. After evaluation of the expressed QconCAT, this study compares transferrin levels obtained by synthetic peptides versus QconCAT peptides as internal standards. Transferrin levels obtained by both internal standards give overlapping, or nearly overlapping, uncertainty values and are near ≈200 mg/dL of transferrin in human serum. Close agreement between the two methods suggests that the quantitative values are reasonable. Using QconCAT and synthetic peptides in parallel gives a refined focus on method development, and the resulting methods should be applicable to other clinically relevant proteins.

In-source decay during matrix-assisted laser desorption/ionization combined with the collisional process in an FTICR mass spectrometer.

The type of ions detected after in-source decay (ISD) in a MALDI source differs according to the ion source pressure and on the mass analyzer used. We present the mechanism leading to the final ISD ions for a Fourier transform-ion cyclotron resonance mass spectrometer (FTICR MS). The MALDI ion source was operated at intermediate pressure to cool the resulting ions and increase their lifetime during the long residence times in the FTICR ion optics. This condition produces not only c', z', and w fragments, but also a, y', and d fragments. In particular, d ions help to identify isobaric amino acid residues present near the N-terminal amino acid. Desorbed ions collide with background gas during desorption, leading to proton mobilization from Arg residues to a less favored protonation site. As a result, in the case of ISD with MALDI FTICR, the influence of the Arg residue in ISD fragmentation is less straightforward than for TOF MS and the sequence coverage is thus improved. MALDI-ISD combined with FTICR MS appears to be a useful method for sequencing of peptides and proteins including discrimination of isobaric amino acid residues and site determination of phosphorylation. Additionally we also used new software for in silico elimination of MALDI matrix peaks from MALDI-ISD FTICR mass spectra. The combination of high resolving power of an FTICR analyzer and matrix subtraction software helps to interpret the low m/z region of MALDI-ISD spectra. Finally, several of these developed methods are applied in unison toward a MALDI ISD FTICR imaging experiment on mouse brain to achieve better results.

MALDI in-source decay, from sequencing to imaging.

Matrix-assisted laser desorption/ionization (MALDI) is now a mature method allowing the identification and, more challenging, the quantification of biopolymers (proteins, nucleic acids, glycans, etc). MALDI spectra show mostly intact singly charged ions. To obtain fragments, the activation of singly charged precursors is necessary, but not efficient above 3.5 kDa, thus making MALDI MS/MS difficult for large species. In-source decay (ISD) is a prompt fragmentation reaction that can be induced thermally or by radicals. As fragments are formed in the source, precursor ions cannot be selected; however, the technique is not limited by the mass of the analyzed compounds and pseudo MS3 can be performed on intense fragments. The discovery of new matrices that enhance the ISD yield, combined with the high sensitivity of MALDI mass spectrometers, and software development, opens new perspectives. We first review the mechanisms involved in the ISD processes, then discuss ISD applications like top-down sequencing and post-translational modifications (PTMs) studies, and finally review MALDI-ISD tissue imaging applications.

Raman spectroscopy and laser desorption mass spectrometry for minimal destructive forensic analysis of black and color inkjet printed documents.

Inkjet ink analysis is the best way to discriminate between printed documents, or even though more difficult, to connect an inkjet printed document with a brand or model of printers. Raman spectroscopy and laser desorption mass spectrometry (LDMS) have been demonstrated as powerful tools for dyes and pigments analysis, which are ink components. The aim of this work is to evaluate the aforementioned techniques for inkjet inks analysis in terms of discriminating power, information quality, and nondestructive capability. So, we investigated 10 different inkjet ink cartridges (primary colors and black), 7 from the HP manufacturer and one each from Epson, Canon and Lexmark. This paper demonstrates the capabilities of three methods: Raman spectroscopy, LDMS and MALDI-MS. Raman spectroscopy, as it is preferable to try the nondestructive approach first, is successfully adapted to the analysis of color printed documents in most cases. For analysis of color inkjet inks by LDMS, we show that a MALDI matrix (9-aminoacridine, 9AA) is needed to desorb and to ionize dyes from most inkjet inks (except Epson inks). Therefore, a method was developed to apply the 9AA MALDI matrix directly onto the piece of paper while avoiding analyte spreading. The obtained mass spectra are very discriminating and lead to information about ink additives and paper compositions. Discrimination of black inkjet printed documents is more difficult because of the common use of carbon black as the principal pigment. We show for the first time the possibility to discriminate between two black-printed documents coming from different, as well as from the same, manufacturers. Mass spectra recorded from black inks in positive ion mode LDMS detect polyethylene glycol polymers which have characteristic mass distributions and end groups. Moreover, software has been developed for rapid and objective comparison of the low mass range of these positive mode LDMS spectra which have characteristic unknown peaks.

An analytical pipeline for MALDI in-source decay mass spectrometry imaging.

In-source decay (ISD) fragmentation as combined with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry allows protein sequencing directly from mass spectra. Acquisition of MALDI-ISD mass spectra from tissue samples is achieved using an appropriate MALDI matrix, such as 1,5-diaminonaphthalene (DAN). Recent efforts have focused on combining MALDI-ISD with mass spectrometry imaging (MSI) to provide simultaneous sequencing and localization of proteins over a thin tissue surface. Successfully coupling these approaches requires the development of new data analysis tools, but first, investigating the properties of MALDI-ISD as applied to mixtures of protein standards reveals a high sensitivity to the relative protein ionization efficiency. This finding translates to the protein mixtures found in tissues and is used to inform the development of an analytical pipeline for data analysis in MALDI-ISD MS imaging, including software to identify the most pertinent spectra, to sequence protein mixtures, and to generate ion images for comparison with tissue morphology. The ability to simultaneously identify and localize proteins is demonstrated by using the analytical pipeline on three tissue sections from porcine eye lens, resulting in localizations for crystallins and cytochrome c. The variety of protein identifications provided by MALDI-ISD-MSI between tissue sections creates a discovery tool, and the analytical pipeline makes this process more efficient.

The modified-bead stretched sample method: development and application to MALDI-MS imaging of protein localization in the spinal cord.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has been used to create spatial distribution maps from lipids, peptides, and proteins in a variety of biological tissues. MALDI-MSI often involves trade-offs between the extent of analyte extraction and desired spatial resolution, compromises that can adversely affect detectability. For example, increasing the extraction time can lead to unwanted analyte spatial redistribution. With the stretched sample method (SSM), the extraction period can be extended, resulting in reduced analyte redistribution while suppressing detection of cationic salt adducts. The SSM involves thaw-mounting a thin tissue section onto a substrate of small glass beads embedded in Parafilm M and then stretching the membrane to fragment the tissue into thousands of bead-sized pieces. Here, we applied the SSM method to MALDI-MSI using rat spinal cord as a model. We used surface-modified beads coated with trypsin or chymotrypsin in order to facilitate controlled digestion and detection of proteins. The enzymatic reactions were maintained by repeatedly condensing water on the stretched sample surface. As a result, new peptides formed by tryptic or chymotryptic protein digestion were detected and identified using a combination of MALDI-MSI and offline liquid chromatography tandem mass spectrometric analysis. Localization of these peptides indicated the distribution of their proteins of origin, including myelin basic protein, actin beta, and tubulin alpha chain. Additionally, we used uncoated beads to create distribution maps of many endogenous lipids and small peptides. The extension of the SSM using modified beads resulted in the creation of mosaic bead surfaces where adjacent beads were coated with different enzymes or other reactive chemicals, permitting investigation of the distributions of a wider range of analytes in biological samples within a single experiment.

MALDI mass spectrometry imaging of neuronal cell cultures.

Mass spectrometry imaging (MSI) provides the ability to detect and identify a broad range of analytes and their spatial distributions from a variety of sample types, including tissue sections. Here we describe an approach for probing neuropeptides from sparse cell cultures using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MSI--at single cell spatial resolution-in both MS and tandem MS modes. Cultures of Aplysia californica neurons are grown on an array of glass beads embedded in a stretchable layer of Parafilm M. As the membrane is stretched, the beads/neurons are separated physically and the separated beads/neurons analyzed via MALDI TOF MS. Compared with direct MS imaging of samples, the stretching procedure enhances analyte extraction and incorporation into the MALDI matrix, with negligible analyte spread between separated beads. MALDI tandem MSI using the stretched imaging approach yields localization maps of both parent and fragment ions from Aplysia pedal peptide, thereby confirming peptide identification. This methodology represents a flexible platform for MSI investigation of a variety of cell cultures, including functioning neuronal networks.

Mass spectrometry imaging using the stretched sample approach.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) can determine tissue localization for a variety of analytes with high sensitivity, chemical specificity, and spatial resolution. MS image quality typically depends on the MALDI matrix application method used, particularly when the matrix solution or powder is applied directly to the tissue surface. Improper matrix application results in spatial redistribution of analytes and reduced MS signal quality. Here we present a stretched sample imaging protocol that removes the dependence of MS image quality on the matrix application process and improves analyte extraction and sample desalting. First, the tissue sample is placed on a monolayer of solid support beads that are embedded in a hydrophobic membrane. Stretching the membrane fragments the tissue into thousands of nearly single-cell sized islands, with the pieces physically isolated from each other by the membrane. This spatial isolation prevents analyte transfer between beads, allowing for longer exposure of the tissue fragments to the MALDI matrix, thereby improving detectability of small analyte quantities without sacrificing spatial resolution. When using this method to reconstruct chemical images, complications result from non-uniform stretching of the supporting membrane. Addressing this concern, several computational tools enable automated data acquisition at individual bead locations and allow reconstruction of ion images corresponding to the original spatial conformation of the tissue section. Using mouse pituitary, we demonstrate the utility of this stretched imaging technique for characterizing peptide distributions in heterogeneous tissues at nearly single-cell resolution.

MALDI mass spectrometric imaging using the stretched sample method to reveal neuropeptide distributions in aplysia nervous tissue.

Neuropeptides are a diverse set of complex cell-cell signaling molecules that modulate behavior, learning, and memory. Their spatially heterogeneous distributions, large number of post-translational modifications, and wide range of physiologically active concentrations make their characterization challenging. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometric imaging is well-suited to characterizing and mapping neuropeptides in the central nervous system. Because matrix application can cause peptide migration within tissue samples, application parameters for MALDI typically represent a compromise between attaining the highest signal quality and preserving native spatial distributions. The stretched sample approach minimizes this trade-off by fragmenting the tissue section into thousands of spatially isolated islands, each approximately 40 mum in size. This inhibits analyte migration between the pieces and, at the same time, reduces analyte-salt adduct formation. Here, we present methodological improvements that enable the imaging of stretched tissues and reveal neuropeptide distributions in nervous tissue from Aplysia californica. The distributions of known neuropeptides are shown to correspond with previous immunohistochemical results, demonstrating that the stretched imaging method is well-suited for working with easily redistributed molecules and heterogeneous tissues and reduces adducts from physiological salts.

Adapting the stretched sample method from tissue profiling to imaging.

The characterization and localization of peptides and proteins in tissues provides information that aids in understanding their function and in characterizing disease states. Over the past decades, the use of MS for the profiling and imaging of biological compounds from tissues has evolved into a powerful modality to accomplish these studies. One recently described sampling approach, the stretched sample method (Monroe, E. B. et al.., Anal. Chem. 2006, 78, 6826-6832), places a tissue section onto an array of glass beads embedded on a Parafilm M membrane. When the membrane is stretched, it separates the tissue section into thousands of cell-sized pieces for tissue profiling by MALDI-MS. The physical separation between beads eliminates analyte redistribution during matrix application and allows long analyte extraction periods without loss of spatial resolution. Here, we enhance this sampling approach by introducing algorithms that enable the reconstruction of ion images from these stretched samples. As the first step, a sample-tailored data acquisition method is devised to obtain mass spectra exclusively from the beads, thereby reducing the time, instrument resources, and data handling required for such MS imaging (MSI) experiments. Next, an image reconstruction algorithm matches data acquired from the stretched sample to the initial bead locations. The efficacy of this method is demonstrated using peptide-coated beads with known peptide distributions and appears well-suited to the MSI of heterogeneous tissue samples.

Chapter 13: Imaging of cells and tissues with mass spectrometry: adding chemical information to imaging.

Techniques that map the distribution of compounds in biological tissues can be invaluable in addressing a number of critical questions in biology and medicine. One of the newer methods, mass spectrometric imaging, has enabled investigation of spatial localization for a variety of compounds ranging from atomics to proteins. The ability of mass spectrometry to detect and differentiate a large number of unlabeled compounds makes the approach amenable to the study of complex biological tissues. This chapter focuses on recent advances in the instrumentation and sample preparation protocols that make mass spectrometric imaging of biological samples possible, including strategies for both tissue and single-cell imaging using the following mass spectrometric ionization methods: matrix-assisted laser desorption/ionization, secondary ion, electrospray, and desorption electrospray.

Transfer of multivariate classification models between laboratory and process near-infrared spectrometers for the discrimination of green Arabica and Robusta coffee beans.

Analogous to the situation found in calibration, a classification model constructed from spectra measured on one instrument may not be valid for prediction of class from spectra measured on a second instrument. In this paper, the transfer of multivariate classification models between laboratory and process near-infrared spectrometers is investigated for the discrimination of whole, green Coffea arabica (Arabica) and Coffea canefora (Robusta) coffee beans. A modified version of slope/bias correction, orthogonal signal correction trained on a vector of discrete class identities, and model updating were found to perform well in the preprocessing of data to permit the transfer of a classification model developed on data from one instrument to be used on another instrument. These techniques permitted development of robust models for the discrimination of green coffee beans on both spectrometers and resulted in misclassification errors for the transfer process in the range of 5-10%.

NeuroPred: a tool to predict cleavage sites in neuropeptide precursors and provide the masses of the resulting peptides.

NeuroPred is a web application designed to predict cleavage sites at basic amino acid locations in neuropeptide precursor sequences. The user can study one amino acid sequence or multiple sequences simultaneously, selecting from several prediction models and optional, user-defined functions. Logistic regression models are trained on experimentally verified or published cleavage data from mollusks, mammals and insects, and amino acid motifs reported to be associated with cleavage. Confidence interval limits of the probabilities of cleavage indicate the precision of the predictions; these predictions are transformed into cleavage or non-cleavage events according to user-defined thresholds. In addition to the precursor sequence, NeuroPred accepts user-specified cleavage information, providing model accuracy statistics based on observed and predicted cleavages. Neuropred also computes the mass of the predicted peptides, including user-selectable post-translational modifications. The resulting mass list aids the discovery and confirmation of new neuropeptides using mass spectrometry techniques. The NeuroPred application, manual, reference manuscripts and training sequences are available at http://neuroproteomics.scs.uiuc.edu/neuropred.html.

Bridging neuropeptidomics and genomics with bioinformatics: Prediction of mammalian neuropeptide prohormone processing.

Neuropeptides are an important class of cell to cell signaling molecules that are difficult to predict from genetic information because of their large number of post-translational modifications. The transition from prohormone genetic sequence information to the determination of the biologically active neuropeptides requires the identification of the cleaved basic sites, among the many possible cleavage sites, that exist in the prohormone. We report a binary logistic regression model trained on mammalian prohormones that is more sensitive than existing methods in predicting these processing sites, and demonstrate the application of this method to mammalian neuropeptidomic studies. By comparing the predictive abilities of a binary logistic model trained on molluscan prohormone cleavages with the reported model, we establish the need for phyla-specific models.