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Exosomes (small extracellular vesicles) in living organisms play an important role in processes such as cell proliferation or intercellular communication. Recently, exosomes have been extensively investigated for biomarker discoveries for various diseases. An important aspect of exosome analysis involves the development of enrichment methods that have been introduced for successful isolation of exosomes. These methods include ultracentrifugation, size exclusion chromatography, polyethylene glycol‐based precipitation, immunoaffinity‐based enrichment, ultrafiltration, and asymmetric flow field‐flow fractionation among others. To confirm the presence of exosomes, various characterization methods have been utilized such as Western blot analysis, atomic force microscopy, electron microscopy, optical methods, zeta potential, visual inspection, and mass spectrometry. Recent advances in high‐resolution separations, high‐performance mass spectrometry and comprehensive proteome databases have all contributed to the successful analysis of exosomes from patient samples. Herein we review various exosome enrichment methods, characterization methods, and recent trends of exosome investigations using mass spectrometry‐based approaches for biomarker discovery. [ABSTRACT FROM AUTHOR]

26 Lin, Z., Simeone, D.M., Anderson, M.A., Brand, R.E., Xie, X., Shedden, K.A., Ruffin, M.T., and Lubman, D.M. Use of non-porous reversed-phase high-performance liquid chromatography for protein profiling and isolation of proteins induced by temperature variations for Siberian permafrost bacteria with identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and capillary electrophoresis-electrospray ionization mass spectrometry. 46 Nie, S., Yin, H., Tan, Z., Anderson, M.A., Ruffin, M.T., Simeone, D.M., and Lubman, D.M. 76 Zhu, J., He, J., Liu, Y., Simeone, D.M., and Lubman, D.M. [Extracted from the article]

Rationale: Exosomes contain biomarkers such as proteins and lipids that help in understanding normal physiology and diseases. Lipids, in particular, are infrequently studied using matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry (MS) for biomarker discovery. In this study, MALDI was equipped with a high‐resolution MS to investigate exosomal lipids from human serum. Methods: Exosomal lipids were profiled using MALDI with Fourier‐transform ion cyclotron resonance (FTICR)‐MS. Four matrices (i.e., α‐cyano‐4‐hydroxycinnamic acid [CHCA], 2,5‐dihydroxybenzoic acid, sinapinic acid, and graphene oxide [GO]) and three sample preparation methods (i.e., dried droplet, thin layer, and two layer) were compared for the number of lipid species detected and the relative abundance of each lipid from human serum and human serum exosomes. Results: In sum, 172 and 89 lipid species were identified from human serum and human serum exosomes, respectively, using all the methods. The highest number of exosome lipid species, 69, was detected using the CHCA matrix, whereas only 8 exosome lipid species were identified using the GO matrix. Among the identified lipid species, phosphatidylcholine was identified most frequently, probably due to the use of a positive ion mode. Conclusions: Exosomes and human serum showed comparable lipid profiles as determined using MALDI‐FTICR‐MS. These findings provide a new perspective on exosomal lipidomics analysis and may serve as a foundation for future lipidomics‐based biomarker research using MALDI‐FTICR‐MS. [ABSTRACT FROM AUTHOR]

Immobilized lectin chromatography can be employed for glycoprotein enrichment, but commonly used columns have limitations of yield and resolution. To improve efficiency and to make the technique applicable to minimal sample material, we have developed a nanoscale chelating Concanavalin A (Con A) monolithic capillary prepared using GMA-EDMA (glycidyl methacrylate-coethylene dimethacrylate) as polymeric support. Con A was immobilized on Cu(II)-charged iminodiacetic acid (IDA) regenerable sorbents by forming a IDA:Cu(II):Con A sandwich affinity structure that has high column capacity, as well as stability. When compared with conventional Con A lectin chromatography, the monolithic capillary enabled the better reproducible detection of over double the number of unique N-glycoproteins in human urine samples. Utility for analysis of minimal biological samples was confirmed by the successful elucidation of glycoprotein profiles in mouse urine samples at the microliter scale. The improved efficiency of the nanoscale monolithic capillary will impact the analysis of glycoproteins in complex biological samples, especially where only limited material may be available.

Protein glycosylation has been implicated in key biological processes including immunological recognition, cellular adhesion, protein folding, and signaling as well as disease progression. Although several methods are available to assess glycosylation of protein structures, none of them is able to screen complex biological samples at a global as well as an individual scale. A novel strategy presented here uses an all-liquid phase enrichment and prefractionation methodology coupled to glycoprotein microarray technology using a multiple lectin-based, biotin--streptavidin detection scheme. Selective detection of glycan structures was made possible by employing multiple lectins to screen glycoprotein standards as well as serum samples from normal subjects or patients with chronic pancreatitis or pancreatic cancer. Interestingly, in some instances, a greater degree of glycosylation was seen in proteins that were underexpressed based on the reversed-phase chromatogram alone. Studies with standard proteins established the limits of detection to be in the 2.5-5-fmol range. Studies on serum samples showed differences in glycosylation patterns, particularly with respect to sialylation, mannosylation, and fucosylation, in normal, pancreatitis, and cancer sera. By coupling glycoprotein enrichment and fractionation with a microarray platform, we have shown that naturally occurring glycoproteins from human serum can be screened and characterized for different glycan structures, thereby allowing one to do comparative studies that monitor individual glycosylation changes within a glycoproteome representing different biological states. This approach may be useful to identify potential biomarkers in cancer.

A method is developed to integrate a protein separation by monolithic capillary reversed-phase high-performance liquid chromatography to on-probe tryptic digestion for subsequent analyses by MALDI-TOF MS and MALDITOF/TOF MS. The method provides a means of directly interfacing separations to MALDI-MS, reducing the amount of time required for traditional procedures involving in-solution enzymatic digestion and sample cleanup prior to MALDI-MS analysis. When used with pI-based fractionation as a first dimension, it provides a means of analyzing complex mixtures of proteins with minimal sample handling and cleanup. The use of monolithic capillary columns sufficiently resolved intact proteins so that peptide mass fingerprinting analysis by MALDI-TOF MS resulted in the identification of close to 40 unique proteins from 120 ng of sample obtained from a prefractionated MCF10 cell line at pH 6.34, where the identifications of several of these proteins were also confirmed by intact MW and tandem mass spectrometric analysis. The reproducibility of this method has been demonstrated to be sufficient for the purpose of protein identifications. Experimental values of protein intact MW are obtained and compared to that expected for each protein identified.

A combination of protein microarrays and two-dimensional liquid-phase separation of proteins has been used for global profiling of the phosphoproteome in human breast cancer cells. This method has been applied to study changes in phosphorylation profile resulting from treatment of the cancer cells with PD173074, a known receptor tyrosine kinase inhibitor. The proteins separated by 2-D liquid-phase separation were arrayed on epoxycoated glass slides and first screened for phosphorylation using fluorescent Pro-Q Diamond stain. The candidate proteins were then identified using MALDI/ESI MS/MS analysis. Further, validation was achieved by immunobiot analysis using anti-phosphotyrosine antibodies. A dynamic range of ~100 was achieved on the microarray when [beta]-casein was used as a standard protein for obtaining quantitative data. Importantly, the power of this method lies in its ability to identify a large group of proteins in a single experiment that are coregnlated in their posttranslational modifications, upon treatment with the inhibitor. Since proteins are known to form interacting circuits that eventually lead to various signaling events, detection of such global phosphorylation profiles might enable delineation of functional pathways that play an important role during cancer initiation and progression.