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Tryptophan (Trp)-catabolic enzymes (TCEs) produce metabolites that activate the aryl hydrocarbon receptor (AHR) and promote tumor progression and immunosuppression in glioblastoma. As therapies targeting TCEs or AHR become available, a better understanding of Trp metabolism is required. Methods: The combination of LC-MS/MS with chemical isobaric labeling enabled the simultaneous quantitative comparison of Trp and its amino group-bearing metabolites in multiple samples. We applied this method to the sera of a cohort of 43 recurrent glioblastoma patients and 43 age- and sex-matched healthy controls. Tumor volumes were measured in MRI data using an artificial neural network-based approach. MALDI MSI visualized Trp and its direct metabolite N-formylkynurenine (FK) in glioblastoma tissue. Analysis of scRNA-seq data was used to detect the presence of Trp metabolism and AHR activity in different cell types in glioblastoma. Results: Compared to healthy controls, glioblastoma patients showed decreased serum Trp levels. Surprisingly, the levels of Trp metabolites were also reduced. The decrease became smaller with more enzymatic steps between Trp and its metabolites, suggesting that Trp availability controls the levels of its systemic metabolites. High tumor volume associated with low systemic metabolite levels and low systemic kynurenine levels associated with worse overall survival. MALDI MSI demonstrated heterogeneity of Trp catabolism across glioblastoma tissues. Analysis of scRNA-seq data revealed that genes involved in Trp metabolism were expressed in almost all the cell types in glioblastoma and that most cell types, in particular macrophages and T cells, exhibited AHR activation. Moreover, high AHR activity associated with reduced overall survival in the glioblastoma TCGA dataset.Conclusion: The novel techniques we developed could support the identification of patients that may benefit from therapies targeting TCEs or AHR activation

The design and synthesis of a proline-based reporter isobaric Tandem Mass Tag structure (TMTpro) is presented. An analysis is made of the performance of the new TMTpro tags in comparison with the current commercially available dimethylpiperidine-reporter-based TMT10/11 reagents. The new reporter structure provides a set of 16 tags for use with resolution of 6.3 mDa mass differences in high resolution mass spectrometers and a set of 9 reagents with 1 Da spacing between reporter ions for single dalton analysis using 9 heavy nuclei per tag. We show similar performance in terms of peptide identification rates and quantification between the TMTpro 16-plex and TMT10/11-plex reagents. We also demonstrate the suitability of the TMTpro reagents for phosphopeptide analysis. The ability to pool 16 samples reduces the overall amount of sample required for each channel, and we anticipate that TMTpro reagents will be a useful enhancement for any protocol that benefits from sample pooling and should reduce missing data.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The lack of biomarkers for early diagnosis, clinical stratification and to monitor treatment response has hampered the development of new therapies for amyotrophic lateral sclerosis (ALS), a clinically heterogeneous neurodegenerative disorder with a variable site of disease initiation and rate of progression. To identify new biomarkers and therapeutic targets, two separate proteomic workflows were applied to study the immunological response and the plasma/brain proteome in phenotypic variants of ALS. Conventional multiplex (TMT) proteomic analysis of peripheral blood mononuclear cells (PBMCs) was performed alongside a recently introduced method to profile neuronal-derived proteins in plasma using brain tissue-enhanced isobaric tagging (TMTcalibrator). The combined proteomic analysis allowed the detection of regulated proteins linked to ALS pathogenesis (RNA-binding protein FUS, superoxide dismutase Cu-Zn and neurofilaments light polypeptide) alongside newly identified candidate biomarkers (myosin-9, fructose-bisphosphate aldolase and plectin). In line with the proteomic results, orthogonal immunodetection showed changes in neurofilaments and ApoE in bulbar versus limb onset fast progressing ALS. Functional analysis of significantly regulated features showed enrichment of pathways involved in regulation of the immune response, Rho family GTPases, semaphorin and integrin signalling. Our cross-phenotype investigation of PBMCs and plasma/brain proteins provides a more sensitive biomarker exploratory platform than conventional case-control studies in a single matrix. The reported regulated proteins may represent novel biomarker candidates and potentially druggable targets.

The ability to detect and diagnose Alzheimer's disease (AD) early is an ever pressing issue, and the development of markers of disease progression that are able to distinguish AD patients from normal aging and patients with alternative forms of dementia, is at the center of the issue. Protein markers of disease, or biomarkers, can be used not only to monitor the progression of AD, but also allow identification of patients suitable for potential therapy, and the response to therapy to be monitored. Cerebrospinal fluid protein biomarkers are important in this early AD diagnosis, and three such biomarkers have been extensively studied and are reviewed here. In addition, post translational protein modifications of proteins important in AD pathology are also discussed. If additional biomarkers can be identified and thoroughly understood, potential therapeutic agents can be better designed, and the effects of therapeutic intervention on disease progression can be monitored.

Adenylate kinases (AK; ATP+AMP double left right arrow 2 ADP; E.C. 2.7.4.3.) are enzymes essentially involved in energy metabolism and macromolecular biosynthesis. As we reported previously, the malarial parasite Plasmodium falciparum possesses one genuine AK and one GTP-AMP phosphotransferase. Analysis of the P. falciparum genome suggested the presence of one additional adenylate kinase, which we designated AK2. Recombinantly produced AK2 was found to be a monomeric protein of 33 kDa showing a specific activity of 10U/mg with ATP and AMP as a substrate pair and to interact with the AK-specific inhibitor P-1,P-5 -(diadenosine-5')-pentaphosphate (IC50 = 200 nM). At its N-terminus AK2 carries a predicted myristoylation sequence. This sequence is only present in AK2 of P. falciparum causing the severe tropical malaria and not in other malarial parasites. We heterologously coexpressed AK2 and P.falciparum N-myristoyltransferase (NMT) in the presence of myristate in Escherichia coli. As demonstrated by protein purification and mass spectrometry, AK2 is indeed myristoylated under catalysis of the parasites' transferase. The modification significantly enhances the stability of the kinase. Furthermore, AK2 and NMT were shown to interact strongly with each other forming a heterodimeric protein in vitro. To our knowledge this is the first direct evidence that P. falciparum NMT myristoylates an intact malarial protein. (C) 2008 Elsevier B.V. All rights reserved.

Raman microspectroscopy was applied for an in situ localization of the malaria pigment hemozoin in Plasmodium falciparum-infected erythrocytes. The Raman spectra (lambdaexc=633 nm) of hemozoin show very intense signals with a very good signal-to-noise ratio. These in situ Raman signals of hemozoin were compared to Raman spectra of extracted hemozoin, of the synthetic analogue beta-hematin, and of hematin and hemin. beta-Hematin was synthesized according to the acid-catalyzed dehydration of hematin and the anhydrous dehydrohalogenation of hemin which lead to good crystals with lengths of about 5-30 microm. The Raman spectra (lambdaexc=1064 nm) of hemozoin and beta-hematin show almost identical behaviors, while some low wavenumber modes might be used to distinguish between the morphology of differently synthesized beta-hematin samples. The intensity pattern of the resonance Raman spectra (lambdaexc=568 nm) of hemozoin and beta-hematin differ significantly from those of hematin and hemin. The most striking difference is an additional band at 1655 cm(-1) which was only observed in the spectra of hemozoin and beta-hematin and cannot be seen in the spectra of hematin and hemin. Raman spectra of the beta-hematin dimer were calculated ab initio (DFT) for the first time and used for an assignment of the experimentally derived Raman bands. The calculated atomic displacements provide valuable insight into the most important molecular vibrations of the hemozoin dimer. With help from these DFT calculations, it was possible to assign the Raman band at 1655 cm(-1) to a mode located at the propionic acid side chain, which links the hemozoin dimers to each other. The Raman band at 1568 cm(-1), which has been shown to be influenced by an attachment of the antimalarial drug chloroquine in an earlier study, could be assigned to a C=C stretching mode spread across one of the porphyrin rings and is therefore expected to be influenced by a pi-pi-stacking to the drug.

The mammalian thioredoxin system, comprising the selenoenzyme thioredoxin reductase (TrxR) and the 12-kDa protein thioredoxin (Trx), is implicated in thiol-mediated antioxidant defense and redox regulatory processes including transcriptional control, DNA synthesis, and apoptosis. Cell proliferation supported by the thioredoxin system can be suppressed by TrxR inhibition. In this study, we assessed the effects of the potent hTrxR inhibitors 4-mercaptopyridine (4′-chloro-2,2′:6′,2ʺ-terpyridine)platinum nitrate (I232N) and 2-mercaptopyridine (4′-chloro-2,2′:6′,2ʺ-terpyridine)platinum nitrate (I252N) on glioblastoma in a rat model. These compounds show no or little cross-resistance with cisplatin and are thus of great clinical interest. Triple intravenous application of 25–35 mg/kg of the compounds led to a significant decrease of tumor growth as determined by magnetic resonance imaging. Metabolic as well as redox parameters in the blood of the animals were not altered. However, TrxR activity was significantly decreased in the tumor tissue, and redox parameters—including glutathione concentrations, total antioxidant status, and the activities of different antioxidant enzymes—showed tissue-specific variations. As indicated by different apoptotic markers, the antitumor activity of I232N is not mediated by the induction of programmed cell death but rather by hTrxR inhibition and DNA intercalation leading to cell cycle arrest.

The homodimeric flavoprotein glutathione reductase (GR) is a central player of cellular redox metabolism, connecting NADPH to the large pool of redox-active thiols. In this work, the inhibition of human GR by a novel gold-phosphole inhibitor (GoPI) has been studied in vitro. Two modes of inhibition are observed, reversible inhibition that is competitive with GSSG followed by irreversible inhibition. When approximately 1 nm GoPI is incubated with NADPH-reduced GR (1.4 nm) the enzyme becomes 50% inhibited. This appears to be the most potent stable inhibitor of human GR to date. Analyzing the monophasic oxidative half-reaction of reduced GR with GSSG at pH 6.9 revealed a K(d)((app)) for GSSG of 63 microm, and a k((obs)max) of 106 s(-1) at 4 degrees C. The reversible inhibition by the gold-phosphole complex [{1-phenyl-2,5-di(2-pyridyl)phosphole}AuCl] involves formation of a complex at the GSSG-binding site of GR (K(d) = 0.46 microm) followed by nucleophilic attack of an active site cysteine residue that leads to covalent modification and complete inactivation of the enzyme. Data from titration spectra, molecular modeling, stopped-flow, and steady-state kinetics support this theory. In addition, covalent binding of the inhibitor to human GR was demonstrated by mass spectrometry. The extraordinary properties of the compound and its derivatives might be exploited for cell biological studies or medical applications, e.g. as an anti-tumor or antiparasitic drug. Preliminary experiments with glioblastoma cells cultured in vitro indicate an anti-proliferative effect of the inhibitor in the lower micromolar range.

Here, we establish a methodology for large-scale quantitative proteomics using SIL (stable isotope labeling) to examine protein expression changes in trophozoite stages of the malaria parasite Plasmodium falciparum following drug treatment. For this purpose, exposure to (13)C6 (15)N1-isoleucine was optimized in order to obtain 99% atomic enrichment. Proteome fractionation with anion exchange chromatography was used to reduce sample complexity and increase quantitative coverage of protein expression. Tryptic peptides of subfractions were subjected to SCX/RP separation, measured by LC-MS/MS, and quantified using the software tool Census. In drug-treated parasites, we identified a total number of 1,253 proteins, thus increasing the overall number of proteins so far identified in the trophozoite stage by 30% in the previous literature. A relative quantification was obtained for more than 800 proteins. About 5% of proteins showed a clear up- or downregulation upon drug treatment.

This chapter summarizes the currently available knowledge on sources of oxidative and nitrosative stress in malarial parasites, the different available detoxification pathways, and the impact on mechanisms of drug action. Plasmodium-infected red blood cells (Plasmodium IRBCs) appear to be under exogenous and endogenous oxidative stress. Malaria parasites induce oxidative stress in their host red blood cell. In the membrane of P. falciparum-parasitized cells, increasing amounts of hemichromes and band 3 aggregates have been demonstrated. Peroxidized IRBCs generate 4-hydroxyalk- 2-enals and alka-2,4-dienals, and these aldehydes are toxic to P. falciparum in vitro. Thus, the antioxidant capabilities of the parasite and RBC are of considerable significance. The majority of the peroxide-detoxifying capacity, however, seems to be provided by peroxiredoxins. Glutathione S-transferase exhibits glutathione peroxidase activity, which might contribute to the total peroxide-reducing capacity of the parasite, since the enzyme is present at very high concentrations. Superoxide dismutase (SOD) is the major enzyme involved in catabolizing the superoxide anion, resulting in production of molecular oxygen and hydrogen peroxide. P. falciparum possesses a classical 2-Cys glutaredoxin (PfGrx1) and a redox-active 1-Cys glutaredoxin-like protein (PfGLP-1). The glyoxalase system consists of glyoxalase I (GloI), glyoxalase II (GloII), and the coenzyme glutathione. It is a cyclic metabolic pathway removing toxic 2-oxoaldehydes like methylglyoxal by converting them to the corresponding nontoxic 2-hydroxycarboxylic acids like D-lactate. Artemisinin has been shown to react with glutathione (GSH) and to increase levels of lipid peroxidation.

Peripheral blood mononuclear cells (PBMCs) are an easy accessible cellular part of the blood organ and, along with platelets, represent the only site of active gene expression in blood. These cells undergo immunophenotypic changes in various diseases and represent a peripheral source of monitoring gene expression and posttranslational modifications relevant to many diseases. Little is known about the source of many blood proteins and we hypothesise that release from PBMCs through active and passive mechanisms may account for a substantial part of the plasma proteome. The use of state-of-the-art proteomic profiling methods in PBMCs will enable minimally invasive monitoring of disease progression or response to treatment and discovery of biomarkers. To achieve this goal, detailed mapping of the PBMC proteome using a sensitive, robust, and quantitative methodological setup is required. We have applied an indepth gel-free proteomics approach using tandem mass tags (TMT), unfractionated and SCX fractionated PBMC samples, and LC-MS/MS with various modulations. This study represents a benchmark in deciphering the PBMC proteome as we provide a deep insight by identifying 4129 proteins and 25503 peptides. The identified proteome defines the scope that enables PBMCs to be characterised as cellular major biomarker pool within the blood organ.

Objective:\ud LC-MS/MS phospho-proteomics is an essential technology to help unravel the complex molecular events that lead to and propagate cancer. We have developed a global phospho-proteomic workflow to determine activity of signaling pathways and drug targets in pancreatic cancer tissue for clinical application.\ud \ud Methods:\ud Peptides resulting from tryptic digestion of proteins extracted from frozen tissue of pancreatic ductal adenocarcinoma and background pancreas (n = 12), were labelled with tandem mass tags (TMT 8-plex), separated by strong cation exchange chromatography, then were analysed by LC-MS/MS directly or first enriched for phosphopeptides using IMAC and TiO2, prior to analysis. In-house, commercial and freeware bioinformatic platforms were used to identify relevant biological events from the complex dataset.\ud \ud Results:\ud Of 2,101 proteins identified, 152 demonstrated significant difference in abundance between tumor and non-tumor tissue. They included proteins that are known to be up-regulated in pancreatic cancer (e.g. Mucin-1), but the majority were new candidate markers such as HIPK1 & MLCK. Of the 6,543 unique phosphopeptides identified (6,284 unique phosphorylation sites), 635 showed significant regulation, particularly those from proteins involved in cell migration (Rho guanine nucleotide exchange factors & MRCKα) and formation of focal adhesions. Activator phosphorylation sites on FYN, AKT1, ERK2, HDAC1 and other drug targets were found to be highly modulated (≥2 fold) in different cases highlighting their predictive power.\ud \ud Conclusion:\ud Here we provided critical information enabling us to identify the common and unique molecular events likely contributing to cancer in each case. Such information may be used to help predict more bespoke therapy suitable for an individual case.

Coevolution of the malarial parasite and its human host has resulted in a complex network of interactions contributing to the homeodynamics of the host-parasite unit. As a rapidly growing and multiplying organism, Plasmodium falciparum depends on an adequate antioxidant defense system that is efficient despite the absence of genuine catalase and glutathione peroxidase. Using different experimental approaches, we demonstrate that P. falciparum imports the human redox-active protein peroxiredoxin 2 (hPrx-2, hTPx1) into its cytosol. As shown by confocal microscopy and immunogold electron microscopy, hPrx-2 is also present in the Maurer's clefts, organelles that are described as being involved in parasite protein export. Enzyme kinetic analyses prove that hPrx-2 accepts Plasmodium cytosolic thioredoxin 1 as a reducing substrate. hPrx-2 accounts for roughly 50% of thioredoxin peroxidase activity in parasite extracts, thus indicating a functional role of hPrx-2 as an enzymatic scavenger of peroxides in the parasite. Under chloroquine treatment, a drug promoting oxidative stress, the abundance of hPrx-2 in the parasite increases significantly. P. falciparum has adapted to adopt the hPrx-2, thereby using the host protein for its own purposes.

Terpyridineplatinum(II) complexes (TPCs) efficiently inhibit the proliferation of glioblastoma cells in vitro and have been tested successfully in a rodent glioblastoma model. Apart from intercalation with DNA, the major mechanism of action of TPCs is a very potent and specific interaction with the human selenoprotein thioredoxin reductase (TrxR). TrxR plays a crucial role in cellular redox homeostasis and protection against oxidative damage. In many malignant cells the thioredoxin system is upregulated, promoting tumor growth and progression. Thus, the thioredoxin system has been proposed to be an attractive target for cancer therapy. This study gives the first comprehensive overview of the effects of TPCs on the transcriptome and proteome of glioblastoma cells. We reveal that under TPC treatment, mechanisms countersteering TrxR inhibition are activated in parallel to DNA-damage-responsive pathways. TPC pressure results in long-term compensatory upregulation of TrxR expression. In parallel, p53 is activated, leading to a range of regulations typical for cell-cycle-arrested cells such as upregulation of CDKN1A, induction of GADD45, inhibition of eIF5A maturation, and reduced phosphorylation of stathmin. We also show that TPCs induce endoplasmic reticulum stress, as they activate the unfolded protein response. This profiling study provides a thorough insight into the spectrum of cellular events resulting from specific TrxR inhibition and characterizes the TPC mode of action.

We studied the effects of sulfur-containing chemopreventive agents, including allyl sulfides and isothiocyanates, on human redox networks. Isothiocyanates inhibited isolated redox-active enzymes in a time- and dose-dependent manner. As shown for the most active compound, benzyl isothiocyanate (BITC), on thioredoxin reductase, the inhibition has an initial competitive part (K i=6.1±1.0 μM) followed by a time-dependent irreversible inhibition (k 2=72.8±25.5 M -1 s-1). Also, glutathione reductase and glutathione S-transferase were irreversibly modified by BITC. Sulforaphane led to irreversible inhibition of the studied redox enzymes, but with 5–10 times lower k 2 values. In contrast, allyl sulfides had only moderate effects on the tested enzymes. However, diallyl disulfide was found to react directly with reduced glutathione (k 2=100 M -2 s-1). This reaction might contribute to enhanced oxidative stress and the induction of the selenoprotein glutathione peroxidase as determined on activity and transcript levels. All chemopreventive agents tested induced transcript levels of genes associated with cell cycle arrest and apoptosis. This upregulation was accompanied by a dose-dependent decrease in cell number. Our data indicate that modulation of cellular redox networks is likely to contribute to the effects of sulfur-containing chemopreventive agents.

The resistance of the malarial parasite Plasmodium falciparum to chloroquine represents an emerging problem since neither mode of drug action nor mechanisms of resistance are fully elucidated. We describe a protein expression profiling approach by SELDI-TOF-MS as a useful tool for studying the proteome of malarial parasites. Reproducible and complex protein profiles of the P. falciparum strains K1, Dd2, HB3 and 3D7 were measured on four array types. Hierarchical clustering led to a clear separation of the two major subgroups "resistant" and "sensitive" as well as of the four parasite strains. Our study delivers sets of regulated proteins derived from extensive comparative analyses of 64 P. falciparum protein profiles. A group of 12 peaks reflecting proteome changes under chloroquine treatment and a set of 10 potential chloroquine resistance markers were defined. Three of these regulated peaks were preparatively enriched, purified and identified. They were shown to represent the plasmodial EXP-1 protein, also called circumsporozoite-related antigen, as well as the alpha- and beta- (delta-) chains of human hemoglobin.

Targeted kinase inhibition is a promising treatment modality for melanoma, which has significantly increased available clinical strategies and improved survival outcomes for patients, whose tumors harbor the BRAF V600E mutation. However, responses are transient and multiple mechanisms for drug resistance can contribute to therapeutic escape. Therefore, a novel multiplexed proteomics approach provides an optimal choice for comprehensively dissecting multiple mechanisms of resistance to BRAFi and combination therapy within one experiment. A large-scale quantitative expression proteomics and phosphoproteomics analysis (SysQuant) was carried out on BRAF V600E mutant melanoma cell lines treated with two different clinically relevant kinase inhibitor combinations: 1) BRAFi/MEKi and 2) BRAFi/PI3Ki. Changes in protein expression and phosphorylation in response to each treatment are determined by comparison to vehicle controls. Cell line models all harbor BRAF V600E mutations, but differ in PTEN status (A375 is WT, while WM793 is PTEN null). Briefly, cells were treated with either drug combination (as above) and harvested at 1hr, 6hrs, 24hrs and 48hrs post-treatment. Control cells were treated with DMSO. Samples were lysed, reduced, alkylated and digested with trypsin. Tryptic peptides from each sample were chemically labeled or “barcoded” with TMT-10plex reagents (4 BRAFi/MEKi time points, 4 BRAFi/PI3Ki time points, 1 DMSO control and a pooled reference sample) and combined for LC-MS/MS. After peptide fractionation with strong cation exchange chromatography and phosphopeptide enrichment, LC-MS/MS peptide sequencing and relative quantification was performed using an Orbitrap Fusion mass spectrometer (Thermo). Raw MS data was searched by Proteome Discoverer and analyzed by in-house R-scripts and Perseus statistical software package. Pathway analysis was done in GeneGO (Metacore). The SysQuant workflow identified >9,000 protein groups and >17,000 unique phosphosites per cell line across different treatments. Principal component analysis of the phosphoproteomics data revealed signaling differences across different treatment conditions and drug combinations are mainly driven by BRAFi and the time post-treatment. K-means clustering was also used to examine trends in the data; as an example, this technique could be used to track signaling changes that correlate with reduction and recovery in ERK signaling. The SysQuant approach provides a systems view of global signaling changes occurring in response to drug treatment. The ability to multiplex samples with TMT allows quantitative deduction of protein expression and phosphorylation patterns that are common or unique in different cell lines, time post-treatment and the effect of combination treatments. This proteomics approach ties protein expression and phosphorylation status in response to combination therapy and generates several hypotheses for further testing with the goal of developing novel combination therapy strategies. Citation Format: Ritin Sharma, Inna Fedorenko, Sasa Koncarevic, Vikram Mitra, Stefan Selzer, Gitte Boehm, Ian Pike, Keiran Smalley, John M. Koomen. Functional proteomics elucidates signaling adaptation driven by combination therapy in BRAF mutant melanoma cell line models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-C12.

The intraerythrocytic protozoan parasite Plasmodium falciparum is responsible for more than 500 million clinical cases of tropical malaria annually. Although exposed to high fluxes of reactive oxygen species, Plasmodium lacks the antioxidant enzymes catalase and glutathione peroxidase. Thus, the parasite depends on the antioxidant capacity of its host cell and its own peroxidases. These are fuelled by the thioredoxin system and are considered to represent the major defense line against peroxides. Five peroxidases that act in different compartments have been described in P. falciparum. They include two typical 2-Cys peroxiredoxins (Prx), a 1-Cys Prx, the so-called antioxidant protein (AOP), which is a further Prx acting on the basis of a 1-Cys mechanism, and a glutathione peroxidase-like thioredoxin peroxidase. Because of their central function in redox regulation and antioxidant defense, some of these proteins might represent highly interesting targets for structure-based drug development. In this article we summarize the present knowledge on the thioredoxin and peroxiredoxin metabolism in malaria parasitized red blood cells. We furthermore report novel data on the biochemical and kinetic characterization of different thioredoxins, of AOP, and of the classic 1-Cys peroxiredoxin of P. falciparum.

Resonance Raman spectroscopy was applied for investigating the malaria pigment hemozoin, which is an important target structure of antimalarial drugs. Morphology-sensitive low wavenumber modes of hemozoin were selectively enhanced with help of excitation wavelengths at lambda = 633 nm and lambda = 647 nm. The assignment of the most prominent bands in the Raman spectra at 343 cm(-1) and 368 cm(-1) was assisted by DFT calculations of the hemozoin dimer. The mode at 343 cm(-1) in the Raman spectrum of hemozoin is strongly enhanced with lambda(exc.) = 647 nm and is represented by a combined, symmetric doming mode of the two hematin units in the hemozoin dimer. The enhancement of this vibration is stronger in the resonance Raman spectrum of hemozoin compared with less crystalline beta-hematin. The selective resonance enhancement of the morphology-sensitive Raman modes of hemozoin is caused by absorption bands in the UV-VIS-NIR spectrum. This absorption spectrum of the crystalline malaria pigment hemozoin shows a strong band at 655 nm. Another broad absorption band at 870 nm is the reason for the strong relative resonance enhancement of the mode at 1372 cm(-1) in the Raman spectrum of crystalline hemozoin with lambda(exc.) = 830 nm. In conclusion, resonance Raman micro-spectroscopy with lambda(exc.) = 647 nm was shown to have great potential as an analytical tool to probe the morphology of hematin samples.