Acute phase serum amyloid A in ovarian cancer as an important component of proteome diagnostic profiling

In the context of serum amyloid A (SAA) identification as ovarian cancer marker derived by SELDI‐MS, its serum levels were measured by immunoassay in different stages of ovarian cancer, in benign gynecological tumors, and in healthy controls. In addition, SELDI‐TOF‐MS spectra were obtained by protocol optimized for the SAA peak intensity. SELDI data on small proteins (5.5–17.5 kDa) and SAA immunoassay data were combined with cancer antigen (CA)125 data in order to study the classification accuracy between cancer and noncancer by support vector machine (SVM), logistic regression, and top scoring pair classifiers. Although an addition of SAA immunoassay data to CA125 data did not significantly improve cancer/noncancer discrimination, SVM applied to combined biomarker data (CA125 and SAA immunoassay variables plus 48 SELDI peak variables) yielded the best classification rate (accuracy 95.2% vs. 86.2% for CA125 alone). Notably, most of discriminatory peaks selected by the classifiers have significant correlation with the major known peaks of SAA (11.7 kDa) and transthyretin (13.9 kDa). Acute phase serum amyloid A (A‐SAA) was proved to be an important member of cancer discriminatory protein profile. Among the eight known ovarian cancer SELDI profile components, A‐SAA is the most relevant to molecular pathogenesis of cancer and it has the highest degree of up‐regulation in disease.     

Towards defining the whole salivary peptidome

We report the identification of 2294 peptides/proteins in whole saliva in the mass range between 700 and 16 000 Da by LC‐MS and MS/MS using a MALDI‐TOF/TOF mass spectrometer. Most of the identified peptides/proteins are originated from cellular debris or plasma components while only 26% (n = 607) correspond to salivary peptides/proteins species. In spite of the presence of the major salivary peptides in all samples from the six subjects analyzed, each individual presents a different pattern of fragments, many deriving from the same protein sequence. All our data, in particular the large number of fragments found, suggest high proteolytic activity insight the oral cavity. The analysis of samples by gelatin zymography showed that all saliva donors displayed multiple proteolytic bands, two identified as cathepsin D and G by MS. Analysis of the cleavage site distribution on the main peptide sequences based on contingency tables shows that the predominant cleavages occur between Gln‐Gly or Tyr‐Gly. These cleavages are largely associated with proline‐rich proteins peptides and with histatin 1 and P‐B peptide, respectively. However, depending on the peptide class, different cleavage hits were observed suggesting the presence of a set of proteases acting in different ways according to different peptide sequences. Comparing the number of cleavages involving all residues, it is possible to observe that 44% (±10%) of the observed cleavages in histatin, statherin and P‐B peptide in all individuals may be explained by cathepsin D, suggesting a major role for this enzyme in oral cavity proteolysis.     

Evaluation of a type 1 diabetes serum cohort by SELDI-TOF MS protein profiling

Proteomics analysis of serum from patients with type 1 diabetes (T1D) may lead to novel biomarkers for prediction of disease and for patient monitoring. However, the serum proteome is highly sensitive to sample processing and before proteomics biomarker research serum cohorts should preferably be examined for potential bias between sample groups. SELDI‐TOF MS protein profiling was used for preliminary evaluation of a biological‐bank with 766 serum samples from 270 patients with T1D, collected at 18 different paediatric centers representing 15 countries in Europe and Japan over 2 years (2000–2002). Samples collected 1 (n = 270), 6 (n = 248), and 12 (n = 248) months after T1D diagnosis were grouped across centers and compared. The serum protein profiles varied with collection site and day of analysis; however, markers of sample processing were not systematically different between samples collected at different times after diagnosis. Three members of the apolipoprotein family increased with time in patient serum collected 1, 6, and 12 months after diagnosis (ANOVA, p<0.001). These results support the use of this serum cohort for further proteomic studies and illustrate the potential of high‐throughput MALDI/SELDI‐TOF MS protein profiling for evaluation of serum cohorts before proteomics biomarker research.     

Protein profiling in pathology: analysis and evaluation of 239 frozen tissue biopsies for diagnosis of B-cell lymphomas

Purpose : We determined the potential value of protein profiling of tissue samples by assessing how precise this approach enables discrimination of B‐cell lymphoma from reactive lymph nodes, and how well the profiles can be used for lymphoma classification. Experimental design : Protein lysates from lymph nodes (n=239) from patients with the diagnosis of reactive hyperplasia (n=44), follicular lymphoma (n=63), diffuse large B‐cell lymphoma (n=43), mantle cell lymphoma (n=47), and chronic lymphocytic leukemia/small lymphocytic B‐cell lymphoma (n=42) were analysed by SELDI‐TOF MS. Data analysis was performed by (i) classification and regression tree‐based analysis and (ii) binary and polytomous logistic regression analysis. Results : After internal validation by the leave‐one‐out principle, both the classification and regression tree and logistic regression classification correctly identified the majority of the malignant (87 and 96%, respectively) and benign cases (73 and 75%, respectively). Classification was less successful since approximately one‐third of the cases of each group were misclassified according to the histological classification. However, an additional mantle cell lymphoma case that was misclassified as chronic lymphocytic leukemia/small lymphocytic B‐cell lymphoma initially was identified based on the protein profile. Conclusions and clinical relevance : SELDI‐TOF MS protein profiling allows for reliable identification of the majority of malignant lymphoma cases; however, further validation and testing robustness in a diagnostic setting is needed.     

Automated serum peptide profiling using novel magnetic C18 beads off-line coupled to MALDI-TOF-MS

Serum peptide profiling by MS is an emerging approach for disease diagnosis and biomarker discovery. A magnetic bead‐based method for off‐line serum peptide capture coupled to MALDI‐TOF‐MS has been recently introduced. However, the reagents are not available to the general scientific community. Here, we developed a protocol for serum peptide capture using novel magnetic C18 beads, and automated the procedure on a high‐throughput magnetic particle processor. We investigated bead equilibration, peptide binding and peptide elution conditions. The method is evaluated in terms of peaks counts and reproducibility of ion intensities in control serum. Overall, the DynaBead‐RPC18‐based serum sample processing protocol reported here is reproducible, robust and allows for the detection of ?200 peptides at m/z 800–4000 of serum that was allowed to clot for 1 h. The average intra‐experiment %CV of normalized ion intensities for crude serum and 0.5% TFA/0.15% n‐octyl glucoside‐treated serum, respectively, were 12% (range 2–38%) and 10% (3–21%) and the inter‐experiment %CVs were 24% (10–53%) and 31% (10–59%). Importantly, this method can be used for serum peptide profiling by anyone in possession of a MALDI‐TOF instrument. In conjunction with the KingFisher® 96, the whole serum peptide capture procedure is high‐throughput (?20 min per isolation of 96 samples in parallel), thereby facilitating large‐scale disease profiling studies.     

Detection of colorectal adenoma and cancer based on transthyretin and C3a-desArg serum levels

Colorectal cancer is the second leading cause of cancer death, and it develops from benign colorectal adenomas in over 95% of patients. Early detection of these cancer precursors by screening tests and their removal can potentially eradicate more than 95% of colorectal cancers before they develop. To discover sensitive and specific biomarkers for improvement of pre‐clinical diagnosis of colorectal adenoma and cancer, we analysed in two independent studies (n = 87 and n = 83 patients) serum samples from colorectal cancer (stage III), colorectal adenoma and control patients using SELDI‐TOF‐MS. Extensive statistical analysis was performed to establish homogeneous patient groups based on their clinical data. Two biomarkers that were each able to distinguish control patients from either colorectal adenoma or colorectal cancer patients (p<0.001) were identified as transthyretin (pre‐albumin) and C3a‐desArg by MS/MS and were further validated by antibody‐based assays (radial immunodiffusion, ELISA). A combination of both proteins clearly indicated the presence of colorectal adenoma or carcinoma. Using a cut‐off of <0.225 g/L for transthyretin and >1974 ng/mL for C3a‐desArg, we found a sensitivity and specificity for colorectal adenoma of 96% and 70%, respectively.     

Cover Picture: Proteomics – Clinical Applications 9/2008

In this issue of Proteomics – Clinical Applications you will find the following highlighted articles: Always probing for more: prostate biomarkers It feels a bit like the late nineteenth century, but instead of a gold rush every two to five years, it's a new favorite target in the biomarker rushes. (Actually, gold rushes go back to ancient Egypt. Biomarkers don't go that far but medical research does.) Here, Burgess et al. take a walk outside the box when they encounter the asymmetry of protein abundance. Rather than synthetically trapping compounds to expose or capture low abundance compounds, they use nature's own: in particular, alpha‐2‐macroglobulin (A2M). A2Ms normal function is to bind proteins that are to be protected from proteolysis, a universal protease inhibitor. Using immunoprecipitation of A2M and comparing cases vs. controls, enhanced levels of heat shock protein 90 in serum was their most interesting candidate for this year's marker rush. Burgess, E. F. et al., Proteomics Clin. Appl. 2008, 2, 1223–1233. Brainwashing samples No, we are not suggesting 1984‐style re‐education to improve proteome productivity. Rather, Dean et al. are reporting on the efficiency of fractionation of brain tissue proteins by graduated detergent extraction prior to 2‐DE. Another anticipated benefit is increased relative concentration of the less abundant proteins. Samples from two areas of the human brain (Brodmann's Area 9 (BA9) and caudate nucleus and putamen CP) were prepared with a sequential extraction kit and compared by 1‐DE and Western blots, 2‐DE and MALDI‐TOF. The conclusion was that no detergent conditions were found that resolved proteins completely but that each detergent point gave a different 2‐D pattern, a benefit for those looking for distinguishing marks. Dean, B. et al., Proteomics Clin. Appl. 2008, 2, 1281–1289. Liver and kidney pie In orthotopic (“full replacement”) liver transplants, one of the most common complications is chronic kidney (yes, kidney) disease. Currently, kidney complications are tracked by functional tests, like serum urea and creatinine levels. If things look suspicious, glomerular filtration rates can be checked. O'Riordan et al. applied SELDI TOF‐MS techniques to serum samples to look for easier, more accurate targets. Serum samples were collected repeatedly over a 6‐month period. Each was divided into six fractions by elution pH or by organic solvent, then examined on weak cation exchange (CM10), hydrophobic (H50) and immobilized metal affinity surfaces (IMAC30). CM10 was best at distinguishing case from control using three proteins and reporting a sensitivity of ～87–94%. On the basis of peptide LC‐MS and 1‐D SDS‐PAGE and confirmed by ELISA, the best single indicator was APO‐AI. Most cases of kidney disease appeared to be linked to the use of calcineurin inhibitors for immune suppression. O'Riordan, A. et al., Proteomics Clin. Appl. 2008, 2, 1338–1348.     

In this issue: Proteomics – Clinical Applications 9/2008

In this issue of Proteomics – Clinical Applications you will find the following highlighted articles: Always probing for more: prostate biomarkers It feels a bit like the late nineteenth century, but instead of a gold rush every two to five years, it's a new favorite target in the biomarker rushes. (Actually, gold rushes go back to ancient Egypt. Biomarkers don't go that far but medical research does.) Here, Burgess et al. take a walk outside the box when they encounter the asymmetry of protein abundance. Rather than synthetically trapping compounds to expose or capture low abundance compounds, they use nature's own: in particular, alpha‐2‐macroglobulin (A2M). A2Ms normal function is to bind proteins that are to be protected from proteolysis, a universal protease inhibitor. Using immunoprecipitation of A2M and comparing cases vs. controls, enhanced levels of heat shock protein 90 in serum was their most interesting candidate for this year's marker rush. Burgess, E. F. et al., Proteomics Clin. Appl. 2008, 2, 1223–1233. Brainwashing samples No, we are not suggesting 1984‐style re‐education to improve proteome productivity. Rather, Dean et al. are reporting on the efficiency of fractionation of brain tissue proteins by graduated detergent extraction prior to 2‐DE. Another anticipated benefit is increased relative concentration of the less abundant proteins. Samples from two areas of the human brain (Brodmann's Area 9 (BA9) and caudate nucleus and putamen CP) were prepared with a sequential extraction kit and compared by 1‐DE and Western blots, 2‐DE and MALDI‐TOF. The conclusion was that no detergent conditions were found that resolved proteins completely but that each detergent point gave a different 2‐D pattern, a benefit for those looking for distinguishing marks. Dean, B. et al., Proteomics Clin. Appl. 2008, 2, 1281–1289. Liver and kidney pie In orthotopic (“full replacement”) liver transplants, one of the most common complications is chronic kidney (yes, kidney) disease. Currently, kidney complications are tracked by functional tests, like serum urea and creatinine levels. If things look suspicious, glomerular filtration rates can be checked. O'Riordan et al. applied SELDI TOF‐MS techniques to serum samples to look for easier, more accurate targets. Serum samples were collected repeatedly over a 6‐month period. Each was divided into six fractions by elution pH or by organic solvent, then examined on weak cation exchange (CM10), hydrophobic (H50) and immobilized metal affinity surfaces (IMAC30). CM10 was best at distinguishing case from control using three proteins and reporting a sensitivity of ～87–94%. On the basis of peptide LC‐MS and 1‐D SDS‐PAGE and confirmed by ELISA, the best single indicator was APO‐AI. Most cases of kidney disease appeared to be linked to the use of calcineurin inhibitors for immune suppression. O'Riordan, A. et al., Proteomics Clin. Appl. 2008, 2, 1338–1348.     

Identification of Apolipoprotein AI as a serum biomarker of chronic kidney disease in liver transplant recipients, using proteomic techniques

Chronic kidney disease (CKD) is a common complication post‐orthotopic liver transplantation (OLT). Development of CKD is detected by monitoring serum urea and creatinine, however disease can occasionally be at an advanced stage before they become abnormal. Therefore, more accurate parameters are required. In order to identify novel biomarkers of CKD, serum was obtained from 47 OLT recipients with CKD (glomerular filtration rate <60 mL/min) and 23 with normal renal function (glomerular filtration rate >90 mL/min). Using the proteomic technique SELDI‐TOF‐MS, three protein biomarkers (55.6 kDa, 9.5 kDa and 11.4 kDa) were identified that, together, could stratify patients into cases or controls with a sensitivity and specificity of 93.6 and 91.3%, respectively. The area under the curve was 0.94. The primary splitter of the groups at 55.6 kDa was an alternative version of a molecule at 27.8 kDa, which was subsequently identified by 1‐D SDS‐PAGE and LC‐ESI‐MS/MS to be Apolipoprotein AI. Protein expression was shown to be reduced in CKD, by both ELISA (p = 0.057) and Western blot analysis (p = 0.003). Apolipoprotein AI is a novel, accurate marker of CKD post‐OLT. It does require further validation in a large, more diverse patient population but could potentially improve detection of CKD.     

A search for novel markers of alcohol abuse using magnetic beads and MALDI-TOF/TOF mass spectrometry

Objective markers are required to assess excessive alcohol consumption, which can lead to a various medical and social problems. In this study, we carried out serum peptidome analyses using the ClinProt^™ system, which consists of magnetic beads and MALDI-TOF/TOF MS, to find novel biomarkers of alcohol abuse in 16 chronic alcoholic patients that were hospitalized for a rehabilitation program. A total of 22 peaks were found to be significantly altered during abstinence. Out of these 22 peaks, 3 peaks that had an m/z of 3000 or less and substantial peak intensities were subjected to MS/MS analysis followed by a MASCOT search. The 1466 Da and the 1616 Da peptides were upregulated on admission and were identified as fragments of fibrinopeptide A and phosphorylated fibrinopeptide A, respectively. On the other hand, the 2660 Da peptide, which was downregulated on admission and increased during abstinence, was identified as a fragment of the fibrinogen α C chain. These peaks were not detectable by the SELDI-TOF MS ProteinChip^® system analysis. The alterations in these peaks induced by alcohol abuse were also seen in γ glutamyltransferase nonresponders. These protein fragments may be additional biomarkers for excessive alcohol drinking.     

Cancer biomarker discovery via low molecular weight serum proteome profiling – Where is the tumor?

Time‐course analyses of rapidly processed serum performed in parallel by SELDI and nanoscale LC‐MS/MS have revealed the temporal correlation of several literature‐based disease markers with ex vivo driven events such that their in vivo existence in healthy subjects is questionable. Identification by MS/MS reveals these putative biomarkers to be byproducts of the coagulation cascade and platelet activation and suggests plasmatic analysis may be preferred. In a pilot plasmatic study, a cohort of naïve prostate cancer (PCa) samples were uniformly distinguished from their age‐matched controls (n = 20) on the basis of multiple peptidic components; most notably by a derivative of complement C₄ at 1863 m/z (GLEEELQFSLGSKINVK, C4_1353–1369). The fully tryptic nature of this and other putative PCa discriminants is consistent with the cleavage specificity of common blood proteases and questions the need for tumor‐derived proteolytic activities as has been proposed. In light of the known correlation of disregulated hemostasis with malignant disease, we suggest the underlying differentiating phenomena in these types of analyses may lie in the temporal disparity of sample activation such that the case (patient) samples are preactivated while the control samples are not.     

Towards stable diagnostic setups in clinical proteomics: Absolute quantitation of peptide biomarkers using MALDI‐TOF‐MS

In routine clinical diagnostics, peptide biomarkers are most commonly quantified using immunological techniques but these methods often lack sensitivity and/or specificity. Hence, quantitative mass spectrometry detection is desirable as an alternative diagnostic tool. To date, quantitative mass spectrometry is mostly based on ESI‐MS coupled to LC, requiring highly sophisticated instrumentation and knowledge and is time consuming and expensive. In contrast, MALDI‐TOF‐MS is a very simple, sensitive and rapid method for the detection of peptide biomarkers. However, the infeasibility of absolute quantification has been a tremendous handicap to the use of MS in stable clinical diagnostics. Here, we describe the development of a technical platform based on ClinProt particles and heavy‐isotope internal peptide standards for the fast and reliable preparation of samples. This combines the advantages of MALDI‐TOF as a read‐out system with absolute quantitation of peptide biomarkers. As a proof‐of‐concept, this platform was successfully employed for the absolute determination of the concentration of the highly abundant serum peptide des‐Ala‐Fibrinopeptide A in 45 serum samples from healthy donors. Such technology essentially contributes to the development of a stable MALDI‐TOF‐MS‐based clinical assay.     

Assessment by Matrix‐Assisted Laser Desorption/Ionization Time‐of‐Flight Mass Spectrometry of the Effects of Preanalytical Variables on Serum Peptidome Profiles Following Long‐Term Sample Storage

Purpose

Human serum and plasma are often used as clinical specimens in proteomics analyses, and peptidome profiling of human serum is a promising tool for identifying novel disease‐associated biomarkers. Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) is widely used for peptidomic biomarker discovery. Careful sample collection and handling are required as either can have a profound impact on serum peptidome patterns, yet the effects of preanalytical variables on serum peptidome profiles have not been completely elucidated. The present study investigated the effects of preanalytical variables, including storage temperature, duration (up to 12 months), and thawing methods, on MALDI‐TOF MS‐based serum peptidome patterns.

Experimental design

Aliquots of serum samples were pretreated with weak cation exchanger magnetic beads using an automated ClinProtRobot system and then analyzed by MALDI‐TOF MS.

Results

A number of significant differences in peak intensities were observed depending on sample processing variables.

Conclusions and clinical relevance

These peaks can be used as sample quality markers to assess the effects of long‐term storage on serum peptidome profiles using MALDI‐TOF MS.

     

Proteomic Helicobacter pylori biomarkers discriminative of low-grade gastric MALT lymphoma and duodenal ulcer

To date no reliable diagnostic method exists to predict, among the very large and clinically heterogeneous group of Helicobacter pylori‐infected patients, the extremely small group at risk for developing low‐grade gastric MALT lymphoma (LG‐MALT). Search of proteomic biomarkers holds promise for the classification of the H. pylori strains with regard to this severe clinical outcome. In the present study 69 H. pylori strains isolated from patients with two different H. pylori‐associated diseases, duodenal ulcer (DU, n=29) and LG‐MALT (n=40) were used. Protein expression patterns of the strains were analyzed by using the high‐throughput methodology SELDI. Selected proteins were purified by means of chromatographic and electrophoretic methods in view of further sequencing by LC‐MS/MS. Univariate analysis (Mann–Whitney test) of the protein expression patterns generated nine significant biomarkers that can discriminate between H. pylori strains from patients with DU and LG‐MALT. These biomarkers are of low molecular weight, ranging from 6 to 26.6 kDa. Among them, two are overexpressed in LG‐MALT strains and seven – in DU strains. Two biomarker proteins, one overexpressed in LG‐MALT strains (13.2 kDa) and another one – overexpressed in DU strains (26.6 kDa), were purified to homogeneity and identified by using LC‐MS/MS as a 50S ribosomal protein L7/L12 and a urease subunit, respectively. These biomarkers can be included in novel protein arrays for the differential diagnosis of H. pylori‐associated clinical outcomes.     

Thymosin β4 is differentially expressed in the cerebrospinal fluid of Creutzfeldt‐Jakob disease patients: a MALDI‐TOF MS protein profiling study

MALDI‐TOF protein profiling analysis permits the detection of peptides and small proteins in complex protein mixtures with great accuracy. We applied this analysis to cerebrospinal fluid (CSF) from 15 patients affected by Creutzfeldt‐Jakob disease (CJD). We compared the levels of the normalized ion signals of 11 sporadic and 4 genetic CJD forms with those from ten healthy control subjects and eight non‐CJD relapsing‐remitting multiple sclerosis patients. In so doing, we detected 61 differentially expressed ion signals in CJD samples compared to controls. Among the 61 signals, 3 signals had significantly increased levels with high statistical significance (p <0.0001) and were located at 3238.3 m/z, 4963.7 m/z, and 8565.3 m/z. We characterized the 5.0 and 8.6 kDa proteins as thymosin β4 N‐acetylated and free ubiquitin, respectively, while the 3.2‐kDa peptide remained uncharacterized. Although elevated ubiquitin levels have previously been described in CJD, we have demonstrated for the first time the involvement of thymosin β4 in a neurodegenerative disease. To support the validity of thymosin β4 levels obtained by MALDI‐TOF analysis, an independent enzyme immunoassay analysis was performed. Moreover, a validation cohort consisting of CSF from three CJD patients, five healthy subjects, and six non‐CJD relapsing‐remitting multiple sclerosis patients was analyzed in a similar way, yielding superimposable results. We propose that thymosin β4 is a potential new candidate marker for the ante mortem diagnosis of CJD disease.     

Translating N‐Glycan Analytical Applications into Clinical Strategies for Ovarian Cancer

Protein glycosylation, particularly N‐linked glycosylation, is a complex posttranslational modification (PTM), which plays an important role in protein folding and conformation, regulating protein stability and activity, cell–cell interaction, and cell signaling pathways. This review focuses on analytical techniques, primarily MS‐based techniques, to qualitatively and quantitatively assess N‐glycosylation while successfully characterizing compositional, structural, and linkage features with high specificity and sensitivity. The analytical techniques explored in this review include LC–ESI–MS/MS and MALDI time‐of‐flight MS (MALDI‐TOF‐MS), which have been used to analyze clinical samples, such as serum, plasma, ascites, and tissue. Targeting the aberrant N‐glycosylation patterns observed in MALDI–MS imaging (MSI) offers a platform to visualize N‐glycans in tissue‐specific regions. The studies on the intra‐patient (i.e., a comparison of tissue‐specific regions from the same patient) and inter‐patient (i.e., a comparison of tissue‐specific regions between different patients) variation of early‐ and late‐stage ovarian cancer (OC) patients identify specific N‐glycan differences that improve understanding of the tumor microenvironment and potentially improve therapeutic strategies for the clinic.     

Changes of serum‐associated fucosylated glycoproteins and changes in glycosylation of IgA in human cirrhosis

Many modifications in N‐glycosylation have been demonstrated in hepatic cirrhosis. These modifications correspond to an increase of a bisecting core alpha(1,6)‐fucosylated biantennary glycan, an increase in core fucosylation, and the presence of an important population of neutral oligosaccharides in human serum of cirrhotic patients. In this study, a glycoproteomic approach which consists of lectin affinity chromatography, MALDI‐TOF MS for the characterization of N‐glycans released from glycoproteins, one‐ and 2‐D PAGE, electrospray ionization quadrupole ion trap (ESI‐QIT) MS was used to identify serum fucosylated glycoproteins related to cirrhosis. Employing this method, we have shown that IgA is one of the major proteins that is responsible of the glycosylation modifications observed in the serum N‐glycome of cirrhotic patients. To our knowledge, this is the first time that aberrant N‐glycosylation of IgA in cirrhosis is described.     

Specific peptides identified by mass spectrometry in placental tissue from pregnancies complicated by early onset preeclampsia attained by laser capture dissection

Preeclampsia is a common pregnancy‐specific syndrome that is diagnosed by the appearance of both increased blood pressure and proteinuria. Preeclampsia is associated with significant fetal and maternal morbidity and mortality. Although the etiology of preeclampsia is unknown, it is evident that abnormal placentation and trophoblast metabolism plays an important role. We therefore analyzed, identified, and verified specific proteins of villous trophoblast and villous stroma in small numbers of microdissected cells (approximately 125 cells) from seven placentas of women with pregnancies complicated by preeclampsia (cases) and seven uncomplicated pregnancies (controls). Tryptic peptide profiling by MALDI‐TOF MS was used for comparison and identification of significantly expressed peptides. The data were analyzed by ClinProTools (Bruker Daltonics) and by principal component analysis. Subsequently, a subset of placental tissues were homogenized and separated on a NanoLC system to obtain sequencing information (MS/MS spectra). We identified specific peptide patterns in the different cell types: villous stroma and trophoblast cells and differences in these cells of placentas from women with pregnancies complicated by early compared to late onset preeclampsia (<34 and >34 wk gestation, respectively) and controls. Principal component analysis revealed significant differences between the groups. The comparison with placental tissue after preterm delivery with unknown cause revealed that placental peptide patterns in early onset preeclampsia could not be explained by preterm delivery per se. Subsequently, specific, discriminating proteins for early onset preeclampsia compared to controls were identified including calcyclin, surfeit locus protein, and choriomammotropin A precursor. The expression of calcyclin was verified in early onset preeclamptic placental sections by immunohistochemistry. These data suggest that in early onset preeclampsia trophoblastic choriomammotropin regulation is abnormal, possibly through abnormal calcyclin expression and regulation.