Multiple reaction monitoring assays for large-scale quantitation of proteins from 20 mouse organs and tissues.

Mouse is the mammalian model of choice to study human health and disease due to its size, ease of breeding and the natural occurrence of conditions mimicking human pathology. Here we design and validate multiple reaction monitoring mass spectrometry (MRM-MS) assays for quantitation of 2118 unique proteins in 20 murine tissues and organs. We provide open access to technical aspects of these assays to enable their implementation in other laboratories, and demonstrate their suitability for proteomic profiling in mice by measuring normal protein abundances in tissues from three mouse strains: C57BL/6NCrl, NOD/SCID, and BALB/cAnNCrl. Sex- and strain-specific differences in protein abundances are identified and described, and the measured values are freely accessible via our MouseQuaPro database: http://mousequapro.proteincentre.com . Together, this large library of quantitative MRM-MS assays established in mice and the measured baseline protein abundances represent an important resource for research involving mouse models.

Structural Elucidation of Intact Rough-type Lipopolysaccharides Using Field Asymmetric Ion Mobility Spectrometry and Kendrick Mass Defect Plots.

Lipopolysaccharides (LPSs) are a hallmark virulence factor of Gram-negative bacteria. They are complex, structurally heterogeneous mixtures due to variations in number, type, and position of their simplest units: fatty acids and monosaccharides. Thus, LPS structural characterization by traditional mass spectrometry (MS) methods is challenging. Here, we describe the benefits of field asymmetric ion mobility spectrometry (FAIMS) for analysis of an intact R-type lipopolysaccharide complex mixture (lipooligosaccharide; LOS). Structural characterization was performed using Escherichia coli J5 (Rc mutant) LOS, a TLR4 agonist widely used in glycoconjugate vaccine research. FAIMS gas-phase fractionation improved the (S/N) ratio and number of detected LOS species. Additionally, FAIMS allowed the separation of overlapping isobars facilitating their tandem MS characterization and unequivocal structural assignments. In addition to FAIMS gas-phase fractionation benefits, extra sorting of the structurally related LOS molecules was further accomplished using Kendrick mass defect (KMD) plots. Notably, a custom KMD base unit of [Na-H] created a highly organized KMD plot that allowed identification of interesting and novel structural differences across the different LOS ion families, i.e., ions with different acylation degrees, oligosaccharides composition, and chemical modifications. Defining the composition of a single LOS ion by tandem MS along with the organized KMD plot structural network was sufficient to deduce the composition of 181 LOS species out of 321 species present in the mixture. The combination of FAIMS and KMD plots allowed in-depth characterization of the complex LOS mixture and uncovered a wealth of novel information about its structural variations.

Deep proteome coverage advances knowledge of Treponema pallidum protein expression profiles during infection.

Comprehensive proteome-wide analysis of the syphilis spirochete, Treponema pallidum ssp. pallidum, is technically challenging due to high sample complexity, difficulties with obtaining sufficient quantities of bacteria for analysis, and the inherent fragility of the T. pallidum cell envelope which further complicates proteomic identification of rare T. pallidum outer membrane proteins (OMPs). The main aim of the present study was to gain a deeper understanding of the T. pallidum global proteome expression profile under infection conditions. This will corroborate and extend genome annotations, identify protein modifications that are unable to be predicted at the genomic or transcriptomic levels, and provide a foundational knowledge of the T. pallidum protein expression repertoire. Here we describe the optimization of a T. pallidum-specific sample preparation workflow and mass spectrometry-based proteomics pipeline which allowed for the detection of 77% of the T. pallidum protein repertoire under infection conditions. When combined with prior studies, this brings the overall coverage of the T. pallidum proteome to almost 90%. These investigations identified 27 known/predicted OMPs, including potential vaccine candidates, and detected expression of 11 potential OMPs under infection conditions for the first time. The optimized pipeline provides a robust and reproducible workflow for investigating T. pallidum protein expression during infection. Importantly, the combined results provide the deepest coverage of the T. pallidum proteome to date.

Structural Elucidation of Intact Rough-Type Lipopolysaccharides using Field Asymmetric Ion Mobility Spectrometry and Kendrick Mass Defect Plots.

Lipopolysaccharide (LPS) is a hallmark virulence factor of Gram-negative bacteria. It is a complex, structurally heterogeneous mixture due to variations in number, type, and position of its simplest units: fatty acids and monosaccharides. Thus, LPS structural characterization by traditional mass spectrometry (MS) methods is challenging. Here, we describe the benefits of field asymmetric ion mobility spectrometry (FAIMS) for analysis of intact R-type lipopolysaccharide complex mixture (lipooligosaccharide; LOS). Structural characterization was performed using Escherichia coli J5 (Rc mutant) LOS, a TLR4 agonist widely used in glycoconjugate vaccine research. FAIMS gas phase fractionation improved the (S/N) ratio and number of detected LOS species. Additionally, FAIMS allowed the separation of overlapping isobars facilitating their tandem MS characterization and unequivocal structural assignments. In addition to FAIMS gas phase fractionation benefits, extra sorting of the structurally related LOS molecules was further accomplished using Kendrick mass defect (KMD) plots. Notably, a custom KMD base unit of [NaH] created a highly organized KMD plot that allowed identification of interesting and novel structural differences across the different LOS ion families; i.e., ions with different acylation degrees, oligosaccharides composition, and chemical modifications. Defining the composition of a single LOS ion by tandem MS along with the organized KMD plot structural network was sufficient to deduce the composition of 179 LOS species out of 321 species present in the mixture. The combination of FAIMS and KMD plots allowed in-depth characterization of the complex LOS mixture and uncovered a wealth of novel information about its structural variations.

Sample Preparation Methods for Targeted Single-Cell Proteomics.

We compared three cell isolation and two proteomic sample preparation methods for single-cell and near-single-cell analysis. Whole blood was used to quantify hemoglobin (Hb) and glycated-Hb (gly-Hb) in erythrocytes using targeted mass spectrometry and stable isotope-labeled standard peptides. Each method differed in cell isolation and sample preparation as follows: 1) FACS and automated preparation in one-pot for trace samples (autoPOTS); 2) limited dilution via microscopy and a novel rapid one-pot sample preparation method that circumvented the need for the solid-phase extraction, low-volume liquid handling instrumentation and humidified incubation chamber; and 3) CellenONE-based cell isolation and the same one-pot sample preparation method used for limited dilution. Only the CellenONE device routinely isolated single-cells from which Hb was measured to be 540-660 amol per red blood cell (RBC), which was comparable to the calculated SI reference range for mean corpuscular hemoglobin (390-540 amol/RBC). FACSAria sorter and limited dilution could routinely isolate single-digit cell numbers, to reliably quantify CMV-Hb heterogeneity. Finally, we observed that repeated measures, using 5-25 RBCs obtained from N = 10 blood donors, could be used as an alternative and more efficient strategy than single RBC analysis to measure protein heterogeneity, which revealed multimodal distribution, unique for each individual.

Chemoproteomic identification of CO2-dependent lysine carboxylation in proteins.

Carbon dioxide is an omnipresent gas that drives adaptive responses within organisms from all domains of life. The molecular mechanisms by which proteins serve as sensors of CO2 are, accordingly, of great interest. Because CO2 is electrophilic, one way it can modulate protein biochemistry is by carboxylation of the amine group of lysine residues. However, the resulting CO2-carboxylated lysines spontaneously decompose, giving off CO2, which makes studying this modification difficult. Here we describe a method to stably mimic CO2-carboxylated lysine residues in proteins. We leverage this method to develop a quantitative approach to identify CO2-carboxylated lysines of proteins and explore the lysine 'carboxylome' of the CO2-responsive cyanobacterium Synechocystis sp. We uncover one CO2-carboxylated lysine within the effector binding pocket of the metabolic signaling protein PII. CO2-carboxylatation of this lysine markedly lowers the affinity of PII for its regulatory effector ligand ATP, illuminating a negative molecular control mechanism mediated by CO2.

Profiling of antimicrobial metabolites of plant growth promoting Pseudomonas spp. isolated from different plant hosts.

In this study, nine strains of Pseudomonas au rantiaca and P. chlororaphis, and two isolates of Pseudomonas sp.: At1RP4 and RS-1, were characterized for the in-vitro production of secondary metabolites in LB, DMB, and King's B media, and of the genes responsible for the production of antagonistic metabolites. Based on 16S rRNA gene sequence, isolates At1RP4 and RS-1 were identified as strains of P. aeruginosa and P. fluorescens. Five phenazine derivatives comprising phenazine, phenazine-1-carboxylic acid (PCA), 2-hydroxyphenazine-1-carboxylic acid (2-OH-Phz-1-COOH), phenazine-1,6-dicarboxylic acid (Phz-1,6-di-COOH), and 2-hydroxyphenazine (2-OH-Phz) were produced by all strains in all three culture media including DMB, King's B and LB. However, 2,8-dihydroxyphenazine, 6-methylphenazine-1-carboxylic acid, pyrrolnitrin, and the ortho-dialkyl-aromatic acids, were produced by the P. aurantiaca and P. chlororaphis strains. In addition, all strains produced 2-acetamidophenol, pyochelin, and diketopiperazine derivatives in variable amounts in all three culture media used. Highest levels of quorum-sensing signal molecules including PQS, 2-Octyl-3-hydroxy-4(1H)-quinolone, and hexahydro-quinoxaline-1,4-dioxide were recorded for P. aeruginosa At1RP4. Moreover, all strains produced volatile hydrogen cyanide (0.95-6.68 µg/L) and the phytohormone indole-3-acetic acid (0.42-13.9 µM). Production of extracellular lipase and protease was recorded in all pseudomonads, whereas, cellulase production and phosphate solubilization were variable. Genes for hydrogen cyanide and phenazine-1-carboxylic acid were detected in all eleven strains while the gene for pyrrolnitrin biosynthesis was amplified in P. aurantiaca and P. chlororaphis strains. Comparative metabolomic analysis provided detailed insights about the strain-specific metabolites in pseudomonads, and their pseudo-relative quantification in different bacterial growth media to be used as single-strain biofertilizer and biocontrol inoculums. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-020-02585-8.

Concentration Determination of >200 Proteins in Dried Blood Spots for Biomarker Discovery and Validation.

The use of protein biomarkers as surrogates for clinical endpoints requires extensive multilevel validation including development of robust and sensitive assays for precise measurement of protein concentration. Multiple reaction monitoring (MRM) is a well-established mass-spectrometric method that can be used for reproducible protein-concentration measurements in biological specimens collected via microsampling. The dried blood spot (DBS) microsampling technique can be performed non-invasively without the expertise of a phlebotomist, and can enhance analyte stability which facilitate the application of this technique in retrospective studies while providing lower storage and shipping costs, because cold-chain logistics can be eliminated. Thus, precise, sensitive, and multiplexed methods for measuring protein concentrations in DBSs can be used for de novo biomarker discovery and for biomarker quantification or verification experiments. To achieve this goal, MRM assays were developed for multiplexed concentration measurement of proteins in DBSs.The lower limit of quantification (LLOQ) was found to have a median total coefficient of variation (CV) of 18% for 245 proteins, whereas the median LLOQ was 5 fmol of peptide injected on column, and the median inter-day CV over 4 days for measuring endogenous protein concentration was 8%. The majority (88%) of the assays displayed parallelism, whereas the peptide standards remained stable throughout the assay workflow and after exposure to multiple freeze-thaw cycles. For 190 proteins, the measured protein concentrations remained stable in DBS stored at ambient laboratory temperature for up to 2 months. Finally, the developed assays were used to measure the concentration ranges for 200 proteins in twenty same sex, same race and age matched individuals.

Biosecurity risks posed by a large sea-going passenger vessel: challenges of terrestrial arthropod species detection and eradication.

Large sea-going passenger vessels can pose a high biosecurity risk. The risk posed by marine species is well documented, but rarely the risk posed by terrestrial arthropods. We conducted the longest running, most extensive monitoring program of terrestrial arthropods undertaken on board a passenger vessel. Surveillance was conducted over a 19-month period on a large passenger (cruise) vessel that originated in the Baltic Sea (Estonia). The vessel was used as an accommodation facility to house workers at Barrow Island (Australia) for 15 months, during which 73,061 terrestrial arthropods (222 species - four non-indigenous (NIS) to Australia) were collected and identified on board. Detection of Tribolium destructor Uytt., a high-risk NIS to Australia, triggered an eradication effort on the vessel. This effort totalled more than 13,700 human hours and included strict biosecurity protocols to ensure that this and other non-indigenous species (NIS) were not spread from the vessel to Barrow Island or mainland Australia. Our data demonstrate that despite the difficulties of biosecurity on large vessels, stringent protocols can stop NIS spreading from vessels, even where vessel-wide eradication is not possible. We highlight the difficulties associated with detecting and eradicating NIS on large vessels and provide the first detailed list of species that inhabit a vessel of this kind.

Molecular phenotyping of laboratory mouse strains using 500 multiple reaction monitoring mass spectrometry plasma assays.

Mouse is the predominant experimental model for the study of human disease due, in part, to phylogenetic relationship, ease of breeding, and the availability of molecular tools for genetic manipulation. Advances in genome-editing methodologies, such as CRISPR-Cas9, enable the rapid production of new transgenic mouse strains, necessitating complementary high-throughput and systematic phenotyping technologies. In contrast to traditional protein phenotyping techniques, multiple reaction monitoring (MRM) mass spectrometry can be highly multiplexed without forgoing specificity or quantitative precision. Here we present MRM assays for the quantitation of 500 proteins and subsequently determine reference concentration values for plasma proteins across five laboratory mouse strains that are typically used in biomedical research, revealing inter-strain and intra-strain phenotypic differences. These 500 MRM assays will have a broad range of research applications including high-throughput phenotypic validation of novel transgenic mice, identification of candidate biomarkers, and general research applications requiring multiplexed and precise protein quantification.

Proteomic Profiling of Leishmania donovani Promastigote Subcellular Organelles.

To facilitate a greater understanding of the biological processes in the medically important Leishmania donovani parasite, a combination of differential and density-gradient ultracentrifugation techniques were used to achieve a comprehensive subcellular fractionation of the promastigote stage. An in-depth label-free proteomic LC-MS/MS analysis of the density gradients resulted in the identification of ∼50% of the Leishmania proteome (3883 proteins detected), which included ∼645 integral membrane proteins and 1737 uncharacterized proteins. Clustering and subcellular localization of proteins was based on a subset of training Leishmania proteins with known subcellular localizations that had been determined using biochemical, confocal microscopy, or immunoelectron microscopy approaches. This subcellular map will be a valuable resource that will help dissect the cell biology and metabolic processes associated with specific organelles of Leishmania and related kinetoplastids.

Multiplexed MRM-Based Protein Quantitation Using Two Different Stable Isotope-Labeled Peptide Isotopologues for Calibration.

When quantifying endogenous plasma proteins for fundamental and biomedical research - as well as for clinical applications - precise, reproducible, and robust assays are required. Targeted detection of peptides in a bottom-up strategy is the most common and precise mass spectrometry-based quantitation approach when combined with the use of stable isotope-labeled peptides. However, when measuring protein in plasma, the unknown endogenous levels prevent the implementation of the best calibration strategies, since no blank matrix is available. Consequently, several alternative calibration strategies are employed by different laboratories. In this study, these methods were compared to a new approach using two different stable isotope-labeled standard (SIS) peptide isotopologues for each endogenous peptide to be quantified, enabling an external calibration curve as well as the quality control samples to be prepared in pooled human plasma without interference from endogenous peptides. This strategy improves the analytical performance of the assay and enables the accuracy of the assay to be monitored, which can also facilitate method development and validation.

Zero-tolerance biosecurity protects high-conservation-value island nature reserve.

Barrow Island, north-west coast of Australia, is one of the world's significant conservation areas, harboring marsupials that have become extinct or threatened on mainland Australia as well as a rich diversity of plants and animals, some endemic. Access to construct a Liquefied Natural Gas (LNG) plant, Australia's largest infrastructure development, on the island was conditional on no non-indigenous species (NIS) becoming established. We developed a comprehensive biosecurity system to protect the island's biodiversity. From 2009 to 2015 more than 0.5 million passengers and 12.2 million tonnes of freight were transported to the island under the biosecurity system, requiring 1.5 million hrs of inspections. No establishments of NIS were detected. We made four observations that will assist development of biosecurity systems. Firstly, the frequency of detections of organisms corresponded best to a mixture log-normal distribution including the high number of zero inspections and extreme values involving rare incursions. Secondly, comprehensive knowledge of the island's biota allowed estimation of false positive detections (62% native species). Thirdly, detections at the border did not predict incursions on the island. Fourthly, the workforce detected more than half post-border incursions (59%). Similar approaches can and should be implemented for all areas of significant conservation value.

Characterization and Diagnostic Application of Trypanosoma cruzi Trypomastigote Excreted-Secreted Antigens Shed in Extracellular Vesicles Released from Infected Mammalian Cells.

Chagas disease, caused by Trypanosoma cruzi, although endemic in many parts of Central and South America, is emerging as a global health threat through the potential contamination of blood supplies. Consequently, in the absence of a gold standard assay for the diagnosis of Chagas disease, additional antigens or strategies are needed. A proteomic analysis of the trypomastigote excreted-secreted antigens (TESA) associated with exosomal vesicles shed by T. cruzi identified ∼80 parasite proteins, with the majority being trans-sialidases. Mass spectrometry analysis of immunoprecipitation products performed using Chagas immune sera showed a marked enrichment in a subset of TESA proteins. Of particular relevance for diagnostic applications were the retrotransposon hot spot (RHS) proteins, which are absent in Leishmania spp., parasites that often confound diagnosis of Chagas disease. Interestingly, serological screens using recombinant RHS showed a robust immunoreactivity with sera from patients with clinical stages of Chagas ranging from asymptomatic to advance cardiomyopathy and this immunoreactivity was comparable to that of crude TESA. More importantly, no cross-reactivity with RHS was detected with sera from patients with malaria, leishmaniasis, toxoplasmosis, or African sleeping sickness, making this protein an attractive reagent for diagnosis of Chagas disease.

Metabolomic profiling of prostate cancer by matrix assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometry imaging using Matrix Coating Assisted by an Electric Field (MCAEF).

In this work, we combined the use of two MALDI matrices (quercetin and 9-aminoacridine), a recently developed new matrix coating technique - matrix coating assisted by an electric field (MCAEF), and matrix-assisted laser desorption/ionization - Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICRMS) to detect and image endogenous compounds in the cancerous and non-cancerous regions of three human prostate cancer (stage II) tissue specimens. After three rounds of imaging data acquisitions (i.e., quercetin for positive and negative ion detection and 9-aminoacridine for negative ion detection), and metabolite identification, a total of 1091 metabolites including 1032 lipids and 59 other metabolites were routinely detected and successfully localized. Of these compounds, 250 and 217 were only detected in either the cancerous or the non-cancerous regions respectively, although we cannot rule out the presence of these metabolites at concentrations below the detection limit. In addition, 152 of the other 624 metabolites showed differential distributions (p<0.05, t-test) between the two regions of the tissues. Further studies on a larger number of clinical specimens will need to be carried out to confirm this large number of apparently cancer-related metabolites. The successful determination of the spatial locations and abundances of these endogenous biomolecules indicated significant metabolism abnormalities - e.g., increased energy charge and under-expression of neutral acyl glycerides, in the prostate cancer samples. To our knowledge, this work has resulted in MALDI-MS imaging of the largest group of metabolites in prostate cancer thus far and demonstrated the importance of using complementary matrices for comprehensive metabolomic imaging by MALDI-MS. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann.

Multiplexed MRM-based assays for the quantitation of proteins in mouse plasma and heart tissue.

The mouse is the most commonly used laboratory animal, with more than 14 million mice being used for research each year in North America alone. The number and diversity of mouse models is increasing rapidly through genetic engineering strategies, but detailed characterization of these models is still challenging because most phenotypic information is derived from time-consuming histological and biochemical analyses. To expand the biochemists' toolkit, we generated a set of targeted proteomic assays for mouse plasma and heart tissue, utilizing bottom-up LC/MRM-MS with isotope-labeled peptides as internal standards. Protein quantitation was performed using reverse standard curves, with LC-MS platform and curve performance evaluated by quality control standards. The assays comprising the final panel (101 peptides for 81 proteins in plasma; 227 peptides for 159 proteins in heart tissue) have been rigorously developed under a fit-for-purpose approach and utilize stable-isotope labeled peptides for every analyte to provide high-quality, precise relative quantitation. In addition, the peptides have been tested to be interference-free and the assay is highly multiplexed, with reproducibly determined protein concentrations spanning >4 orders of magnitude. The developed assays have been used in a small pilot study to demonstrate their application to molecular phenotyping or biomarker discovery/verification studies.

Multiplexed panel of precisely quantified salivary proteins for biomarker assessment.

An increasingly popular "absolute" quantitative technique involves the SRM or MRM approach with stable isotope-labeled standards (SIS). Using this approach, many proteins in human plasma/serum have been quantified for biomarker assessment and disease stratification. Due to the complexity of plasma and the invasive nature of its collection, alternative biosamples are currently being explored. Here, we present the broadest panel of multiplexed MRM assays with SIS peptides for saliva proteins developed to date. The validated panel consists of 158 candidate human saliva protein biomarkers, inferred from 244 interference-free peptides. The resulting concentrations were reproducibly quantified over a 6 order-of-magnitude concentration range (from 218 µg/mL to 88 pg/mL; average CVs of 12% over analytical triplicates). All concentrations were determined from reverse standard curves, which were generated using a constant concentration of endogenous material with varying concentrations of spiked-in SIS peptides. The large-scale screening of the soluble and membrane-associated proteins contained within the 158-plex assay could present new opportunities for biomarker assessment and clinical diagnostics.

Insights from the pollination drop proteome and the ovule transcriptome of Cephalotaxus at the time of pollination drop production.

BACKGROUND AND AIMS: Many gymnosperms produce an ovular secretion, the pollination drop, during reproduction. The drops serve as a landing site for pollen, but also contain a suite of ions and organic compounds, including proteins, that suggests diverse roles for the drop during pollination. Proteins in the drops of species of Chamaecyparis, Juniperus, Taxus, Pseudotsuga, Ephedra and Welwitschia are thought to function in the conversion of sugars, defence against pathogens, and pollen growth and development. To better understand gymnosperm pollination biology, the pollination drop proteomes of pollination drops from two species of Cephalotaxus have been characterized and an ovular transcriptome for C. sinensis has been assembled. METHODS: Mass spectrometry was used to identify proteins in the pollination drops of Cephalotaxus sinensis and C. koreana RNA-sequencing (RNA-Seq) was employed to assemble a transcriptome and identify transcripts present in the ovules of C. sinensis at the time of pollination drop production. KEY RESULTS: About 30 proteins were detected in the pollination drops of both species. Many of these have been detected in the drops of other gymnosperms and probably function in defence, polysaccharide metabolism and pollen tube growth. Other proteins appear to be unique to Cephalotaxus, and their putative functions include starch and callose degradation, among others. Together, the proteins appear either to have been secreted into the drop or to occur there due to breakdown of ovular cells during drop production. Ovular transcripts represent a wide range of gene ontology categories, and some may be involved in drop formation, ovule development and pollen-ovule interactions. CONCLUSIONS: The proteome of Cephalotaxus pollination drops shares a number of components with those of other conifers and gnetophytes, including proteins for defence such as chitinases and for carbohydrate modification such as ß-galactosidase. Proteins likely to be of intracellular origin, however, form a larger component of drops from Cephalotaxus than expected from studies of other conifers. This is consistent with the observation of nucellar breakdown during drop formation in Cephalotaxus The transcriptome data provide a framework for understanding multiple metabolic processes that occur within the ovule and the pollination drop just before fertilization. They reveal the deep conservation of WUSCHEL expression in ovules and raise questions about whether any of the S-locus transcripts in Cephalotaxus ovules might be involved in pollen-ovule recognition.

The use of matrix coating assisted by an electric field (MCAEF) to enhance mass spectrometric imaging of human prostate cancer biomarkers.

In this work, we combined a newly developed matrix coating technique - matrix coating assisted by an electric field (MCAEF) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to enhance the imaging of peptides and proteins in tissue specimens of human prostate cancer. MCAEF increased the signal-to-noise ratios of the detected proteins by a factor of 2 to 5, and 232 signals were detected within the m/z 3500-37500 mass range on a time-of-flight mass spectrometer and with the sinapinic acid MALDI matrix. Among these species, three proteins (S100-A9, S100-A10, and S100-A12) were only observed in the cancerous cell region and 14 proteins, including a fragment of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 2, a fragment of cAMP-regulated phosphoprotein 19, 3 apolipoproteins (C-I, A-I, and A-II), 2 S100 proteins (A6 and A8), ß-microseminoprotein, tumor protein D52, α-1-acid glycoprotein 1, heat shock protein ß-1, prostate-specific antigen, and 2 unidentified large peptides at m/z 5002.2 and 6704.2, showed significantly differential distributions at the p < 0.05 (t-test) level between the cancerous and the noncancerous regions of the tissue. Among these 17 species, the distributions of apolipoprotein C-I, S100-A6, and S100-A8 were verified by immunohistological staining. In summary, this study resulted in the imaging of the largest group of proteins in prostate cancer tissues by MALDI-MS reported thus far, and is the first to show a correlation between S100 proteins and prostate cancer in a MS imaging study. The successful imaging of the three proteins only found in the cancerous tissues, as well as those showing differential expressions demonstrated the potential of MCAEF-MALDI/MS for the in situ detection of potential cancer biomarkers. Copyright © 2015 John Wiley & Sons, Ltd.