Integrated analysis of the cerebrospinal fluid peptidome and proteome

Cerebrospinal fluid (CSF) is the only body fluid in direct contact with the brain and thus is a potential source of biomarkers. Furthermore, CSF serves as a medium of endocrine signaling and contains a multitude of regulatory peptides. A combined study of the peptidome and proteome of CSF or any other body fluid has not been reported previously. We report confident identification in CSF of 563 peptide products derived from 91 precursor proteins as well as a high confidence CSF proteome of 798 proteins. For the CSF peptidome, we use high accuracy mass spectrometry (MS) for MS and MS/MS modes, allowing unambiguous identification of neuropeptides. Combination of the peptidome and proteome data suggests that enzymatic processing of membrane proteins causes release of their extracellular parts into CSF. The CSF proteome has only partial overlap with the plasma proteome, thus it is produced locally rather than deriving from plasma. Our work offers insights into CSF composition and origin.     

Simultaneous extraction and rapid visualization of peptidomic and lipidomic body fluids fingerprints using mesoporous aluminosilicate and MALDI‐TOF MS

Herein we report the use of mesoporous aluminosilicate (MPAS) for the simultaneous extraction of peptides and lipids from complex body fluids such as human plasma and synovial fluid. We show that MPAS particles, given their mesostructural features with nanometric pore size and high surface area, are an efficient device for simultaneous extraction of peptidome and lipidome from as little as a few microliters of body fluids. The peptides and the lipids, selected and enriched by MPAS particles and rapidly visualized by MALDI‐TOF MS, could form part of a diagnostic profile of the “peptidome” and the “lipidome” of healthy versus diseased subjects in comparative studies. The ability of this approach to rapidly reveal the overall pattern of changes in both lipidome and peptidome signatures of complex biofluids could be of valuable interest for handling large numbers of samples required in ‐omics studies for the purpose of finding novel biomarkers.     

Comprehensive analysis of the N and C terminus of endogenous serum peptides reveals a highly conserved cleavage site pattern derived from proteolytic enzymes

The human serum proteome is closely associated with the state of the body. Endogenous peptides derived from proteolytic enzymes cleaving on serum proteins are widely studied due to their potential application in disease-specific marker discovery. However, the reproducibility of peptidome analysis of endogenous peptides is significantly influenced by the proteolytic enzymes within body fluids, thereby limiting the clinical use of the endogenous peptides. We comprehensively investigated the N and C terminus of endogenous peptides using peptidomics. The cleavage site patterns of the N and C terminus and adjacent sites from all the identified endogenous peptides were highly conserved under different sample preparation conditions, including long-term incubation at 37°C and pretreatment with repeated freeze-thaw cycles. Furthermore, a distinguishable cleavage site pattern was obtained when a different disease serum was analyzed. The conserved cleavage site pattern derived from proteolytic enzymes holds potential in highly specific disease diagnosis.     

Salivary peptidomics

In recent years, there has been an increased interest in the study of saliva. This bodily fluid contains a vast number of protein species, the salivary peptidome, of low molecular weight, comprising approximately 40–50% of the total secreted proteins, in addition to peptides generated by proteolysis of proteins of different sources. Owing to the presence of other components, in particular mucins and enzymes, some distinctive requirements and precautions related to sample collection, time of analysis, sample preservation and treatment are necessary for the successful analysis of salivary peptides. More than 2000 peptides compose the salivary peptidome, from which only 400–600 are directly derived from salivary glands, suggesting an important qualitative peptide contribution of other sources, namely of epithelial cells. Proteolysis events are the main supply for the peptidome and considerable efforts have been made to identify the resulting fragments, the cleavage sites and the involved proteases. The salivary proteins more prone to proteolysis are proline-rich proteins (PRPs; acidic PRPs and basic PRPs), statherin, histatins and P-B peptide. Gln–Gly cleavages are largely associated with PRP classes, while Tyr–Gly cleavages are related to histatin 1 and to the P-B peptide. The interest in saliva has been growing for clinical purposes, as it is an alternative sample to other traditional bodily fluids, such as blood or urine, since it involves an easy and noninvasive collection. In fact, apart from its usefulness as a source of information for the prognosis, diagnosis and treatment of oral diseases, such as Sjögren’s syndrome, gum disease, tooth decay or oral cancer, saliva might also be seen as a potential tool to the diagnosis of systemic diseases. Owing to the enormous amount of previously discovered salivary peptide species, in this article, we attempt to harmonize the nomenclature, following International Union of Pure and Applied Chemistry recommendations.     

Salivary peptidomics

In recent years, there has been an increased interest in the study of saliva. This bodily fluid contains a vast number of protein species, the salivary peptidome, of low molecular weight, comprising approximately 40-50% of the total secreted proteins, in addition to peptides generated by proteolysis of proteins of different sources. Owing to the presence of other components, in particular mucins and enzymes, some distinctive requirements and precautions related to sample collection, time of analysis, sample preservation and treatment are necessary for the successful analysis of salivary peptides. More than 2000 peptides compose the salivary peptidome, from which only 400-600 are directly derived from salivary glands, suggesting an important qualitative peptide contribution of other sources, namely of epithelial cells. Proteolysis events are the main supply for the peptidome and considerable efforts have been made to identify the resulting fragments, the cleavage sites and the involved proteases. The salivary proteins more prone to proteolysis are proline-rich proteins (PRPs; acidic PRPs and basic PRPs), statherin, histatins and P-B peptide. Gln-Gly cleavages are largely associated with PRP classes, while Tyr-Gly cleavages are related to histatin 1 and to the P-B peptide. The interest in saliva has been growing for clinical purposes, as it is an alternative sample to other traditional bodily fluids, such as blood or urine, since it involves an easy and noninvasive collection. In fact, apart from its usefulness as a source of information for the prognosis, diagnosis and treatment of oral diseases, such as Sj?gren's syndrome, gum disease, tooth decay or oral cancer, saliva might also be seen as a potential tool to the diagnosis of systemic diseases. Owing to the enormous amount of previously discovered salivary peptide species, in this article, we attempt to harmonize the nomenclature, following International Union of Pure and Applied Chemistry recommendations.     

Comprehensive peptidome analysis of mouse livers by size exclusion chromatography prefractionation and nanoLC-MS/MS identification

Peptidome analysis has received increasing attention in recent years. Cancer diagnosis by serum peptidome has also been reported by peptides' profiling for discovery of peptide biomarkers. Tissue, which may have a higher biomarker concentration than blood, has not been investigated extensively by means of peptidome analysis. Here, a method for the peptidome analysis of mouse liver was developed by the combination of size exclusion chromatography (SEC) prefractionation with nano-liquid chromatography-tamdem mass spectrometry (nanoLC-MS/MS) analysis. The extracted peptides from mouse liver were separated according to their molecular weight using a size exclusion column. MALDI-TOF MS was used to characterize the molecular weight distribution of the peptides in fractions eluted from the SEC column. The low molecular weight (LMW) (MW < 3000 Da) peptides in the collected fractions were directly analyzed by LC-MS/MS which resulted in the identification of 1181 unique peptides (from 371 proteins). The high molecular weight (HMW) (MW > 3000 Da) peptides in the early two fractions from the SEC column were first digested with trypsin, and the resulted digests were then analyzed by LC-MS/MS, which led to the identification of 123 and 127 progenitor proteins of the HMW peptides in fractions 1 and 2, respectively. Analysis of the peptides' cleavage sites showed that the peptides are cleaved in regulation, which may reflect the protease activity and distribution in body, and also represent the biological state of the tissue and provide a fresh source for biomarker discovery.     

Peptidomic analysis of human reflex tear fluid

Tear fluid is a complex mixture of biological compounds, including carbohydrates, lipids, electrolytes, proteins, and peptides. Despite the physiological importance of tear fluid, little is known about the identity of its endogenous peptides. In this study, we analyzed and identified naturally occurring peptide molecules in human reflex tear fluid by means of LC-MALDI-TOF–TOF. Tandem MS analyses revealed 30 peptides, most of which have not been identified before. Twenty-six peptides are derived from the proline-rich protein 4 and 4 peptides are derived from the polymeric immunoglobulin receptor. Based on their structural characteristics, we suggest that the identified tear fluid peptides contribute to the protective environment of the ocular surface.     

Peptidomics technologies for human body fluids

Peptides play a central role in many physiological processes. In order to analyse comprehensively all peptides and small proteins of a whole organism or a subsystem (peptidome), the use of technologies other than 2D gel electrophoresis is necessary. Approaches that use liquid chromatography or affinity purification and mass spectrometric identification have now been developed and applied successfully to the analysis of human body fluids.     

Peptidomics technologies for human body fluids

Peptides play a central role in many physiological processes. In order to analyse comprehensively all peptides and small proteins of a whole organism or a subsystem (peptidome), the use of technologies other than 2D gel electrophoresis is necessary. Approaches that use liquid chromatography or affinity purification and mass spectrometric identification have now been developed and applied successfully to the analysis of human body fluids.     

A comprehensive peptidome profiling technology for the identification of early detection biomarkers for lung adenocarcinoma

The mass spectrometry-based peptidomics approaches have proven its usefulness in several areas such as the discovery of physiologically active peptides or biomarker candidates derived from various biological fluids including blood and cerebrospinal fluid. However, to identify biomarkers that are reproducible and clinically applicable, development of a novel technology, which enables rapid, sensitive, and quantitative analysis using hundreds of clinical specimens, has been eagerly awaited. Here we report an integrative peptidomic approach for identification of lung cancer-specific serum peptide biomarkers. It is based on the one-step effective enrichment of peptidome fractions (molecular weight of 1,000-5,000) with size exclusion chromatography in combination with the precise label-free quantification analysis of nano-LC/MS/MS data set using Expressionist proteome server platform. We applied this method to 92 serum samples well-managed with our SOP (standard operating procedure) (30 healthy controls and 62 lung adenocarcinoma patients), and quantitatively assessed the detected 3,537 peptide signals. Among them, 118 peptides showed significantly altered serum levels between the control and lung cancer groups (p<0.01 and fold change >5.0). Subsequently we identified peptide sequences by MS/MS analysis and further assessed the reproducibility of Expressionist-based quantification results and their diagnostic powers by MRM-based relative-quantification analysis for 96 independently prepared serum samples and found that APOA4 273-283, FIBA 5-16, and LBN 306-313 should be clinically useful biomarkers for both early detection and tumor staging of lung cancer. Our peptidome profiling technology can provide simple, high-throughput, and reliable quantification of a large number of clinical samples, which is applicable for diverse peptidome-targeting biomarker discoveries using any types of biological specimens.     

Unravelling in vitro variables of major importance for the outcome of mass spectrometry-based serum proteomics

The use of mass spectrometry (MS) for analysing low-molecular weight proteins and peptides from biological fluids has a great, yet not fully realized, potential for biomarker discovery. To prune MS-data as much as possible for non-relevant non-biological variation the development of standardized protocols for handling and processing the samples before MS and adjusting data after MS to compensate for method-induced variability are warranted. This calls for knowledge about how different variables contribute to MS-based proteome analyses. In addition, identification of the peptides involved in pre-analytical variation will be helpful in evaluating the clinical significance of predictive models derived from MS data. Using human sera, extraction by weak cation-exchange magnetic beads, and analysis by MALDI-TOF MS we here evaluated pre-analytical variation and identify peptides involved in this. The influences of humidity, temperature, and time for preparation of sera on spectral changes were evaluated. Also, the reproducibility of the methods and the effect of a baseline correction procedure were examined. Low temperatures, short handling times, and a baseline correction procedure minimize the contribution of artifacts to sample variability as observed by MS. The complement split product C3f and fragments thereof appear to be sensitive indicators of sample handling induced modifications. Other peptides that are indicative of such variability are fibrin and kininogen fragments. Using strict experimental guidelines as well as standardized sample collection procedures it is possible to obtain reproducible peak intensities and positions in serum mass profiling using magnetic bead-based fractionation and MALDI-TOF MS.     

Advances in proximal fluid proteomics for disease biomarker discovery

Although serum/plasma has been the preferred source for identification of disease biomarkers, these efforts have been met with little success, in large part due the relatively small number of highly abundant proteins that render the reliable detection of low abundant disease-related proteins challenging due to the expansive dynamic range of concentration of proteins in this sample. Proximal fluid, the fluid derived from the extracellular milieu of tissues, contains a large repertoire of shed and secreted proteins that are likely to be present at higher concentrations relative to plasma/serum. It is hypothesized that many, if not all, proximal fluid proteins exchange with peripheral circulation, which has provided significant motivation for utilizing proximal fluids as a primary sample source for protein biomarker discovery. The present review highlights recent advances in proximal fluid proteomics, including the various protocols utilized to harvest proximal fluids along with detailing the results from mass spectrometry- and antibody-based analyses.     

Sampling Human Indigenous Saliva Peptidome Using a Lollipop-Like Ultrafiltration Probe: Simplify and Enhance Peptide Detection for Clinical Mass Spectrometry

Although human saliva proteome and peptidome have been revealed ^1-2 they were majorly identified from tryptic digests of saliva proteins. Identification of indigenous peptidome of human saliva without prior digestion with exogenous enzymes becomes imperative, since native peptides in human saliva provide potential values for diagnosing disease, predicting disease progression, and monitoring therapeutic efficacy. Appropriate sampling is a critical step for enhancement of identification of human indigenous saliva peptidome. Traditional methods of sampling human saliva involving centrifugation to remove debris ^3-4 may be too time-consuming to be applicable for clinical use. Furthermore, debris removal by centrifugation may be unable to clean most of the infected pathogens and remove the high abundance proteins that often hinder the identification of low abundance peptidome.Conventional proteomic approaches that primarily utilize two-dimensional gel electrophoresis (2-DE) gels in conjugation with in-gel digestion are capable of identifying many saliva proteins ^5-6. However, this approach is generally not sufficiently sensitive to detect low abundance peptides/proteins. Liquid chromatography-Mass spectrometry (LC-MS) based proteomics is an alternative that can identify proteins without prior 2-DE separation. Although this approach provides higher sensitivity, it generally needs prior sample pre-fractionation ⁷ and pre-digestion with trypsin, which makes it difficult for clinical use. To circumvent the hindrance in mass spectrometry due to sample preparation, we have developed a technique called capillary ultrafiltration (CUF) probes ^8-11. Data from our laboratory demonstrated that the CUF probes are capable of capturing proteins in vivo from various microenvironments in animals in a dynamic and minimally invasive manner ^8-11. No centrifugation is needed since a negative pressure is created by simply syringe withdrawing during sample collection. The CUF probes combined with LC-MS have successfully identified tryptic-digested proteins ^8-11. In this study, we upgraded the ultrafiltration sampling technique by creating a lollipop-like ultrafiltration (LLUF) probe that can easily fit in the human oral cavity. The direct analysis by LC-MS without trypsin digestion showed that human saliva indigenously contains many peptide fragments derived from various proteins. Sampling saliva with LLUF probes avoided centrifugation but effectively removed many larger and high abundance proteins. Our mass spectrometric results illustrated that many low abundance peptides became detectable after filtering out larger proteins with LLUF probes. Detection of low abundance saliva peptides was independent of multiple-step sample separation with chromatography. For clinical application, the LLUF probes incorporated with LC-MS could potentially be used in the future to monitor disease progression from saliva.     

Deciphering the ovarian cancer ascites fluid peptidome

Background

Conventional proteomic approaches have thus far been unable to identify novel serum biomarkers for ovarian cancer that are more sensitive and specific than the current clinically used marker, CA-125. Because endogenous peptides are smaller and may enter the circulation more easily than proteins, a focus on the low-molecular-weight region may reveal novel biomarkers with enhanced sensitivity and specificity. In this study, we deciphered the peptidome of ascites fluid from 3 ovarian cancer patients and 3 benign individuals (ascites fluid from patients with liver cirrhosis).

Results

Following ultrafiltration of the ascites fluids to remove larger proteins, each filtrate was subjected to solid phase extraction and fractionated using strong cation exchange chromatography. The resultant fractions were analyzed using an Orbitrap mass spectrometer. We identified over 2000 unique endogenous peptides derived from 259 proteins. We then catalogued over 777 peptides that were found only in ovarian cancer ascites. Our list of peptides found in ovarian cancer specimens includes fragments derived from the proteins vitronectin, transketolase and haptoglobin.

Conclusions

Peptidomics may uncover previously undiscovered disease-specific endogenous peptides that warrant further investigation as biomarkers for ovarian cancer.     

Prediction of Cell-Penetrating Peptides

Cell-penetrating peptides, CPPs, are used as delivery vectors for pharmacologically interesting substances, such as antisense oligonucleotides, proteins and peptides. We present a general principle for designing cell-penetrating peptides derived from naturally occurring proteins as well as from randomly generated polyamino acid sequences. Thereby, we introduce a novel pharmacological principle for identification of cell-penetrating peptides for which the applications can be numerous, including cellular transduction vectors and mimics of intracellular protein–protein interactions. The methods of identifying a CPP comprises assessing the averaged bulk property values of the defined sequence, and ensuring that they fall within the bulk property value interval obtained from the training set. Despite this simplistic approach, the search criteria proved useful for finding CPP properties in either proteins or random sequences. We have experimentally verified cell-penetrating properties of 10–20-mer peptides derived from naturally occurring proteins as well as from random poly-amino acids. We note that since CPPs can be found in part of the protein sequences that may govern protein interactions, it is possible to produce cell-penetrating protein agonists or antagonists.     

Discovery of Antimicrobial Peptides in Cervical‐Vaginal Fluid from Healthy Nonpregnant Women via an Integrated Proteome and Peptidome Analysis

Cervical‐vaginal fluid (CVF) covers the lower part of the female reproductive system and functions in the homeostasis and immunity of the surrounding tissues. In contrast to the CVF proteome of both nonpregnant and pregnant women, the CVF peptidome has not been reported to date. In the current study, we identified 1087 proteins in CVF, of which 801 proteins were not previously identified in CVF proteomes. The presence of the tissue‐specific proteins oviductal glycoprotein 1 and tubulin polymerization–promoting protein family member 3 strongly suggests that the tissues of the upper female reproductive tract contribute to the protein composition of CVF. The tremendous catalytic potential of CVF was highlighted by the identification of 85 proteases and the detection of pH‐dependent trypsin‐like proteolytic activity. Over 1000 endogenous peptides were detected in the CVF peptidome, and 39 peptides are predicted to have antimicrobial activity. The detailed proteomic and peptidomic analysis of CVF will further aid in the delineation of pathways related to reproduction, immunity and host defense, and assist in developing new biomarkers for malignant and other diseases of the female reproductive tract. Data are available via ProteomeXchange with identifiers PXD004450 (CVF peptidome) and PDX004363 (CVF proteome).     

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.     

Peptidomics of the larval Drosophila melanogaster central nervous system

Neuropeptides regulate most, if not all, biological processes in the animal kingdom, but only seven have been isolated and sequenced from Drosophila melanogaster. In analogy with the proteomics technology, where all proteins expressed in a cell or tissue are analyzed, the peptidomics approach aims at the simultaneous identification of the whole peptidome of a cell or tissue, i.e. all expressed peptides with their posttranslational modifications. Using nanoscale liquid chromatography combined with tandem mass spectrometry and data base mining, we analyzed the peptidome of the larval Drosophila central nervous system at the amino acid sequence level. We were able to provide biochemical evidence for the presence of 28 neuropeptides using an extract of only 50 larval Drosophila central nervous systems. Eighteen of these peptides are encoded in previously cloned or annotated precursor genes, although not all of them were predicted correctly. Eleven of these peptides were never purified before. Eight other peptides are entirely novel and are encoded in five different, not yet annotated genes. This neuropeptide expression profiling study also opens perspectives for other eukaryotic model systems, for which genome projects are completed or in progress.     

The turnover kinetics of major histocompatibility complex peptides of human cancer cells

Peptides presented by the major histocompatibility complex (MHC) are derived from the degradation of cellular proteins. Thus, the repertoire of these peptides (the MHC peptidome) should correlate better with the cellular protein degradation scheme (the degradome) than with the cellular proteome. To test the validity of this statement and to determine whether the majority of MHC peptides are derived from short lived proteins, from defective ribosome products, or from regular long lived cellular proteins we analyzed in parallel the turnover kinetics of both MHC peptides and cellular proteins in the same cancer cells. The analysis was performed by pulse-chase experiments based on stable isotope labeling in tissue culture followed by capillary chromatography and tandem mass spectrometry. Indeed only a limited correlation was observed between the proteome and the MHC peptidome observed in the same cells. Moreover a detailed analysis of the turnover kinetics of the MHC peptides helped to assign their origin to normal, to short lived or long lived proteins, or to the defective ribosome products. Furthermore the analysis of the MHC peptides turnover kinetics helped to direct attention to abnormalities in the degradation schemes of their source proteins. These observations can be extended to search for cancer-related abnormalities in protein degradation, including those that lead to loss of tumor suppressors and cell cycle regulatory proteins.     

A novel peptidomics approach to detect markers of Alzheimer's disease in cerebrospinal fluid

Sensitive and specific diagnosis and monitoring of disease progression are of prime importance to develop new therapies for Alzheimer's disease patients. Although the diagnostic accuracy, verified by pathological examination is high, it is currently not possible to diagnose Alzheimer's disease with a high degree of certainty until relatively late in the disease process. Here, we have undertaken a peptidome analysis of postmortem cerebrospinal fluid of neuropathologically confirmed Alzheimer's disease patients and non-demented controls using a combination of methods and technologies. This includes novel sample preparation based on the enrichment of endogenous, proteolytically derived peptides as well as peptides non-covalently bound to abundant proteins. We observed differences in peptide profiles associated with Alzheimer's disease in the endogenous peptide fraction and in the protein-bound peptide fraction. The discriminating peptides in the unbound peptide fraction were identified as VGF nerve growth factor inducible precursor, and complement C4 precursor, whereas the discriminating peptides in the protein-bound fraction were identified as VGF nerve growth factor inducible precursor, and alpha-2-HS-glycoprotein.