Imaging mass spectrometry: A new tool for pathology in a molecular age

Mass spectrometry (MS) provides unique advantages for the analysis of clinical specimens, and these capabilities have been critical to the advancement of diagnostic medicine. To date, LC-MS is the MS platform most commonly used for diagnostics; however, LC-MS based proteomics is very labor intensive and costly to implement for high volume assays. Furthermore, when analyzing tissue samples, additional laborious sample preparation steps must be employed (e.g. extraction methods or laser microdissection). The direct analysis of cells and tissues by MALDI imaging MS has developed significant momentum for applications that have diagnostic potential. MALDI imaging MS provides molecular information from specific cell types within tissue sections; however, this laser-based approach significantly reduces the analysis time for each location sampled. This Viewpoint discusses the technologies for direct analysis of tissues, the potential for diagnostic applications using MALDI imaging MS, and the challenges faced in the transfer of the technology to the clinical laboratory.     

A peptide-centric approach to breast cancer biomarker discovery utilizing label-free multiple reaction monitoring mass spectrometry

We report an approach for multiplex analysis of cancer biomarkers based on the measurement of diagnostic peptides in whole tissue protein digests. Label-free quantitation with MS3 multiple reaction monitoring (MRM) was developed to afford accurate analysis of prospective marker peptides in a panel of breast tumors. This approach provides an economical and robust alternative to stable isotope-based methods. It is equally applicable to the analysis of samples derived from tissue biopsy, aspirate, or plasma and can be easily translated to clinic.     

Proteomics in clinical prostate research

The incidence of early prostate cancer (PCa) has increased rapidly in recent years. The majority of newly diagnosed PCa are in early tumor phase. Presently, we do not have adequate biomarkers to assess tumor aggressiveness in individual cases. Consequently, too many patients are given curatively intended treatment. An exploration of the human proteome may provide clinically useful markers. 2-DE has been successfully used for analysis of the protein phenotype using clinical samples. Proteins are separated according to size and charge, gels are compared by image analysis, protein spots of interest are excised, and proteins identified by MS. This method is exploratory and allows protein identification. However, low-abundance proteins are difficult to detect and 2-DE is currently too labor-intensive for routine use. In recent years, nongel based techniques, such as LC-MS, SELDI-MS, and protein arrays have emerged. They require smaller sample sizes and can be more automated than 2-DE. In this review, we describe studies of the protein expression of benign prostatic tissue and PCa, which is likely to serve as the first step in prognostic biomarker discovery. The prostate proteome is still far from a complete mapping which would enhance our understanding of PCa biology.     

Reconstructing the pipeline by introducing multiplexed multiple reaction monitoring mass spectrometry for cancer biomarker verification: an NCI-CPTC initiative perspective

Proteomics holds great promise in personalized medicine for cancer in the post-genomic era. In the past decade, clinical proteomics has significantly evolved in terms of technology development, optimization and standardization, as well as in advanced bioinformatics data integration and analysis. Great strides have been made for characterizing a large number of proteins qualitatively and quantitatively in a proteome, including the use of sample fractionation, protein microarrays and MS. It is believed that differential proteomic analysis of high-quality clinical biospecimen (tissue and biofluids) can potentially reveal protein/peptide biomarkers responsible for cancer by means of their altered levels of expression and/or PTMs. Multiple reaction monitoring, a multiplexed platform using stable isotope dilution-MS with sensitivity and reproducibility approaching that of traditional ELISAs commonly used in the clinical setting, has emerged as a potentially promising technique for next-generation high-throughput protein biomarker measurements for diagnostics and therapeutics.     

Development of an immuno tandem mass spectrometry (iMALDI) assay for EGFR diagnosis

The epidermal growth factor receptor (EGFR) is highly expressed in a variety of tumors, and is therefore an important biomarker for cancer diagnosis and a target for cancer therapy. We have developed a novel peptide-based immuno tandem mass spectrometry (iMALDI) diagnostic assay for highly sensitive, highly specific, and quantitative analysis of EGFR, which we have applied to the detection of the EGFR peptide in three cell lines and in a tumor biopsy sample. This assay is capable of detecting the EGFR target peptide bound to the antibody beads at attomole levels. The ability to directly obtain amino acid sequence data by MS/MS on any affinity-captured peptides provides specificity to this diagnostic technique. This avoids the problem of "false positives" which can result from the nonspecific binding that can occur with any affinity-based technique. The addition of stable-labeled versions of the target peptide (synthesized from stable-isotope coded amino acids) as internal standards allows absolute quantitation of the target protein.     

MS analysis of rheumatoid arthritic synovial tissue identifies specific citrullination sites on fibrinogen

Purpose : Citrullination is a post‐translational modification of arginine residues to citrulline catalyzed by peptidyl arginine deiminases. Induced expression of citrullinated proteins are frequently detected in various inflammatory states including arthritis; however, direct detection of citrullination in arthritic samples has not been successfully performed in the past. Experimental design : Citrullination of human fibrinogen, a candidate autoantigen in arthritis, was studied. Accurate identification of citrullinated fibrinogen peptides from rheumatoid arthritis synovial tissue specimens was performed using accurate mass and retention time analysis. Results : A peptide with the sequence ESSSHHPGIAEFPSRGK corresponding to amino acids 559–575 of fibrinogen α‐chain was identified to be citrullinated with an occupancy rate between 1.4 and 2.5%. Citrullination of the peptide KREEAPSLRPAPPPISGGGYRARPAK corresponding to amino acids 52–77 of the fibrinogen β‐chain was identified with an occupancy rate of 1.2%. Conclusions and clinical relevance : We report a proof of principle study for the identification of citrullinated proteins and within them, identification of citrullination sites and quantification of their occupancies in synovial tissue from rheumatoid arthritis patients using high‐resolution MS. Detailed studies on which molecules are citrullinated in arthritis can provide information about their role in immune regulation and serve as novel biomarkers and potentially even as therapeutic targets.     

Proteomic analysis of breast cancer tissue reveals upregulation of actin-remodeling proteins and its relevance to cancer invasiveness

There is an emerging interest in protein expression profiling with the aim of identifying novel diagnostic markers and therapeutic targets in breast cancer. We analyzed breast cancer tissues by 2-D DIGE using a narrow range IPG strip (pH?5.5-6.7) after the immunodepletion of serum albumin and Ig. Sixty-three protein spots were detected with more than ±1.8-fold differences (p?<0.05 for three technical replicates) from a set of tissue samples in which three tumor and three nontumor samples were randomly selected from six breast cancer subjects and pooled separately. Of these, 53 proteins were successfully identified by MS. Among the proteins whose levels were increased, we identified three novel WD-repeat-motif-bearing proteins that have been known to be involved in actin remodeling: Arp2/3 complex subunit 2 (p34-Arc), coronin-1A and WD-repeat protein 1 (Wdr1). Significantly increased amounts of p34-Arc and coronin-1A in breast cancer were also shown by Western blot analysis of matched tumor and nontumor tissue samples (N?=?11, p?<0.05), and were consistent with the mRNA levels retrieved from publicly available microarray databases. The siRNA knockdown of p34-Arc attenuated the invasion of SK-BR3 breast cancer cells into Matrigel. In contrast, the overexpression of coronin-1A increased this invasive activity. Taken together, the cellular levels of p34-Arc and coronin-1A were linked to cancer development and migration. The data obtained from the present study provides new insight into the management of breast cancer.     

Differentially expressed proteins of MCF-7 human breast cancer cells affected by Zilongjin, a complementary Chinese herbal medicine

Purpose : Zilongjin, a complementary Chinese herbal medicine, has been used to alleviate the adverse effects of chemotherapeutic drugs in cancer therapy. However, the mechanisms of anti‐cancer activity of Zilongjin are still largely unkonwn. Experimental design : First, the proteomic approach of combined 2‐DE and ESI‐MS/MS was used to investigate the effect of Zilongjin on the protein expression in MCF‐7 cells. Then, the differential expression of some proteins was confirmed by Western blot, cytoimmunofluoresecnce, and quantitative real‐time RT‐PCR analysis. Results : The identified proteins with differential expression, involved in such events as protein translation, cellular signal transduction, cytoskeleton formation and transportation, include seven downregulating proteins, such as Eukaryotic translation initiation factor 3 subunit I, Eukaryotic translation initiation factor 1A Y‐chromosomal, Ran‐specific GTPase‐activating protein, Ubiquitin‐conjugating enzyme E2 N, Tropomodulin‐3, Macrophage‐capping protein, and Tumor protein D52, as well as two upregulating proteins, HSP β‐1 and keratin18. Moreover, the differential expression of three proteins was confirmed. Conclusions and clinical relevance : (i) These results provide a new insight into the molecular mechanisms of Zilongjin on therapy for breast cancer. (ii) The application of the proteomic approaches will result in the more extended appreciation of Chinese medicine than those known at present.     

Characterisation of tumoral markers correlated with ErbB2 (HER2/Neu) overexpression and metastasis in breast cancer

The receptor tyrosine kinase ErbB2 (HER2/neu) is overexpressed in ?30% of breast cancers and is associated with poor prognosis and an increased likelihood of metastasis. Clinical treatments such as trastuzumab are effective in less than 35% of women diagnosed as ErbB2‐positive, highlighting the necessity of searching for novel targets and alternative therapies. Herein, a proteomic screening strategy combining quantitative‐based gel electrophoresis and MS was used to compare the protein expression of 48 normal human breast and tumour tissues differing in ErbB2 expression and lymph node status. The aim was to identify proteins associated with the aggressive phenotype of ErbB2‐positive breast cancer which could be potential biomarkers of the disease as well as therapy targets. In total, 177 protein isoforms (107 gene products) differentially expressed between tissue groups were identified. Immunohistochemical staining of a tissue‐microarray was used for validation of selected protein candidates. We found that expression of HSP90α, laminin and GSTP1 significantly correlated with ErbB2 expression, while others such as AGR2, NM23H1 and Annexin 2 were overexpressed in greater than 40% of tumours. Finally, knocking‐down the expression by RNA interference of three candidates, AGR2, Transgelin2 and NM23H1 resulted in an enhanced invasive capacity of MDA‐MB435 cells. These data support the involvement of these targets in tumour progression and identify them as novel biomarkers of the disease.     

Mass spectrometry imaging for the proteomic study of clinical tissue

Over the last decade, MALDI-MS imaging has been used by researchers to explore areas of proteomics, lipidomics and metabolomics in samples of clinical origin for both targeted and global biomarker analysis. Numerous technological advancements in MS and clinical tissue MS imaging have been accomplished; hence, in this article we aim to critically discuss whether MS imaging has now in fact become a true champion of the ‘Omics Era’. In order to assess the potential for it to be routinely used in the clinical setting, it is pertinent to discuss some of its limitations, and to examine how these have been addressed by researchers. The key limitations of the technique we will discuss in this viewpoint article are as follows: sample throughput; relevance to patients, the availability of validated/standardised techniques; and integration with conventional pathology and other medical imaging techniques. Good progress has been made over the last 5 years in overcoming these limitations that had previously restricted the use of this technology in the clinical setting.     

An optimum method designed for 2-D DIGE analysis of human arterial intima and media layers isolated by laser microdissection

The formation and progression of atherosclerotic lesions involve complex mechanisms which are still not fully understood. A variety of cell types from the distinct arterial layers are implicated in the whole process from lipid accumulation within the vascular wall to plaque development and final rupture. In the present work, we employ the combination of laser microdissection and pressure catapulting and 2-D DIGE saturation labeling to investigate the human intima and media sub-proteomes isolated from atherosclerotic (coronary and aorta) or non-atherosclerotic vessels (preatherosclerotic coronary arteries). Laser microdissection and pressure catapulting allows the specific isolation of regions of interest. In turn, DIGE saturation labeling overcomes the limitation of extensive microdissection times to recover the protein amount required to perform comparative 2-DE, particularly when dealing with tissue regions rich in myofilament proteins, which result in low protein recovery. The compatibility and optimum performance of both techniques were investigated in detail, paying special attention to tissue staining and protein solubilization. Since scarce amount of protein obtained from microdissected tissue made it impossible to directly perform protein identification from 2-DE spots by MS, we performed in-solution digestion followed by LC-MS/MS analysis of total protein extracts from intima and media in order to get an overall picture of protein composition. Proteins so identified confirm the nature of the isolated regions. Finally, similar spot resolution on 2-D DIGE gels was obtained for the different human artery types (coronary, aorta) and studied layers (intima, media), setting the basis for future clinical comparative studies.     

Decreased annexin A3 expression correlates with tumor progression in papillary thyroid cancer

Purpose : The aim of this study is to identify the potential tumor markers that function in carcinogenesis and tumor progression, thus providing important diagnostic and prognostic information. Experimental design : We performed 2‐D gel electrophoresis and MALDI‐TOF MS to investigate the differentially expressed proteins in 25 papillary thyroid carcinoma tissues. For validation of candidate proteins and investigation of clinical significance, we performed Western, Northern blot analysis and immunohistochemical staining. Results : Our proteomic analyses revealed significantly decreased annexin A3 expression in papillary thyroid carcinoma at both the protein and mRNA levels, compared with normal thyroid tissue. ANXA3 immunoreactivity was not significantly correlated with lymph node metastasis, multifocality, capsular invasion or perithyroidal extension in thyroid cancer. However, the tumor subgroup with a lymph node metastasis score of >3 displayed significantly lower ANXA3 expression than did subgroups with negative and ≤3 scores (p=0.001). Moreover, ANXA3 expression was markedly lower in large tumors (>1 cm in diameter) than in microcarcinomas (p=0.001). Conclusion and clinical relevance : Decreased expression of ANXA3 in papillary thyroid cancer supports the idea that ANXA3 may be an effective marker of microcarcinoma, and a negative predictor of papillary thyroid cancer progression.     

Towards a comprehensive proteome of normal and malignant human colon tissue by 2-D-LC-ESI-MS and 2-DE proteomics and identification of S100A12 as potential cancer biomarker

The aim of this study was to characterize the proteome of normal and malignant colonic tissue. We previously studied the colon proteome using 2‐DE and MALDI‐MS and identified 734 proteins (Roeßler, M., Rollinger, W., Palme S., Hagmann, M.‐L., et al.., Clin. Cancer Res. 2005, 11, 6550–6557). Here we report the identification of additional colon proteins from the same set of tissue samples using a complementary nano‐flow 2‐D‐LC‐ESI‐MS. In total, 484 proteins were identified in colon. Of these, 252 had also been identified by the 2‐DE/MALDI‐MS approach, whereas 232 proteins were unique to the 2‐D‐LC‐ESI‐MS analysis. Comparing protein expression in neoplastic and normal colon tissue indicated elevated expression of several proteins in colorectal cancer, among them the well established tumor marker carcinoembryonic antigen, as well as calnexin, 40S ribosomal protein S15a, serpin H1, and S100A12. Overexpression of these proteins was confirmed by immunoblotting. Serum levels of S100A12 were determined by ELISA and were found to be strongly elevated in colorectal cancer patients compared to healthy individuals. We conclude, that 2‐D‐LC‐ESI‐MS is a powerful approach to identify and compare protein profiles of tissue samples, that it is complementary to 2‐DE/MALDI‐MS approaches and has the potential to identify novel biomarkers.     

Spectral Analysis as a Tool for Financial Policy: An Analysis of the Short-End of the British Term Structure

In this paper, we show how to derive the spectra and cross-spectra of economic time series from an underlying econometric or VAR model. This allows us to conduct a proper frequency analysis evaluation of economic and financial variables on a reduced sample of data, without it being ruled out by the large sample requirements of direct spectral estimation. We show, in particular, how this can be done for time-varying models and time-varying spectra. We use our techniques to show how the behaviour of British interest rates changed during and following the ERM crisis of 1992/3.     

Quantitative proteomic analysis of ovarian cancer cells identified mitochondrial proteins associated with Paclitaxel resistance

Paclitaxel has been widely used as an anti-mitotic agent in chemotherapy for a variety of cancers and adds substantial efficacy as the first-line chemotherapeutic regimen for ovarian cancers. However, the frequent occurrence of paclitaxel resistance limits its function in long-term management. Despite abundant clinical and cellular demonstration of paclitaxel resistant tumors, the molecular mechanisms leading to paclitaxel resistance are poorly understood. Using genomic approaches, we have previously identified an association between a BTB/POZ gene, Nac1, and paclitaxel resistance in ovarian cancer. The experiments presented here have applied multiple quantitative proteomic methods to identify protein changes associated with paclitaxel resistance and Nac1 function. The SKOV-3 ovarian serous carcinoma cell line, which has inducible expression of dominant negative Nac1, was used to determine the paclitaxel treatment associated changes in the presence and absence of functional Nac1. Quantitative proteomic analyses were performed using iTRAQ labeling and mass spectrometry. Two label-free quantitative proteomic methods: LC-MS and spectral count were used to increase confidence of proteomic quantification. A total of 1371 proteins were quantified by at least one of the quantitative proteomic methods. Candidate proteins related to paclitaxel and NAC1 function were identified in this study. Go analysis of the protein changes identified upon paclitaxel resistance revealed that cell component enrichment related to mitochondria. Moreover, tubulin and mitochondrial proteins were the major cellular components with changes associated with paclitaxel treatment. This suggests that mitochondria may play a role in paclitaxel resistance.     

MS characterization of apheresis samples from rheumatoid arthritis patients for the improvement of immunoadsorption therapy - a pilot study

Identification of proteins from apheresis samples was performed by both SDS-PAGE and 2-D gel separation of eluted proteins from staphylococcal protein A-based immunoadsorption columns (Prosorba^®) followed by MS peptide mass fingerprinting and MS/MS peptide sequencing on a MALDI QIT TOF mass spectrometer. MS/MS peptide sequencing was performed in conjunction with a micro reversed phase HPLC configured with an online MALDI plate-spotting device. Apheresis treatment had been performed in three patients with longstanding therapy refractory rheumatoid arthritis. 2-D gels displayed ca. 500 spots representing proteins that were eluted from the Prosorba^® columns. From 54 gels, a total of 1256 protein spots had been picked and yielded in the identification of 56 non-redundant proteins without counting isoforms. Proteins from the eluates belong to five major groups comprising (i) immunoglobulins (IgG, IgA, IgM heavy and light chains; about 40% of the spots), (ii) proteins involved in coagulation, (iii) HDL/LDL-associated proteins, (iv) proteins from the complement system, and (v) acute phase proteins. MS analysis showed that the full-length C3 complement protein had been cleaved upon complement activation, presumably on the column, such that the anaphylatoxin C3a was produced and released during therapy. Our results are consistent with clinical observations on both patient responses to therapy and reported adverse events. For the first time, direct molecular information has become available to support mechanistic reasoning for the principle of function of staphylococcal protein A-based immunoadsorption therapy and for the explanation of adverse events. According to our results, removal and/or modulation of immune complexes together with complement activation can be regarded as the major events that are taking place during Prosorba^® therapy. In order to avoid complement activation and induction of an inflammatory cascade, we suggest the prevention of C3a anaphylatoxin-related reactions during immunoadsorption therapy.     

Proteomics of nipple aspirate fluid, breast cyst fluid, milk, and colostrum

Proteomic analysis of breast fluids has wide-ranging clinical applications. Protein expression in nipple aspirate fluid and breast cyst fluid may prove valuable for the detection and monitoring of breast cancer, but has been hampered by the lack of a single marker with sufficient breast cancer sensitivity and specificity to be clinically useful. The assessment of multiple proteins may offer a more powerful cancer detection tool. Breast cancer is particularly difficult to detect in women who are lactating. The identification of cancer predicting proteins in milk may prove very helpful in an early cancer detection in this group of women. A better understanding of the protein composition of milk and colostrum should improve infant care and nutrition, and lead to alternatives for individuals with milk allergies.     

Phosphoproteomics: A possible route to novel biomarkers of breast cancer

Proteomics is rapidly transforming the way that cancer and other pathologies are investigated. The ability to identify hundreds of proteins and to compare their abundance in different clinical samples presents a unique opportunity for direct identification of novel disease markers. Furthermore, recent advances allow us to analyse and compare PTMs. This gives an additional dimension for defining a new class of protein biomarker based not only on abundance and expression but also on the occurrence of covalent modifications specific to a disease state or therapy response. Such modifications are often a consequence of the activation/inactivation of a particular disease related pathway. In this review we evaluate the available information on breast cancer related protein-phosphorylation events, illustrating the rationale for investigating this PTM as a target for breast cancer research with eventual clinical relevance. We present a critical survey of the published experimental strategies to study protein phosphorylation on a system wide scale and highlight recent specific advances in breast cancer phosphoproteomics. Finally we discuss the feasibility of establishing novel biomarkers for breast cancer based on the detection of patterns of specific protein phosphorylation events.     

Microdispensing and Nanovial Arrays Provides Rapid Automated Protein/Peptide Identification using MALDI-TOF MS

The rapid development in the field of microtechnology has opened up new possibilities of rapid screening protocols in pharmaceutical research and drug development. Today there is a strong need for high-throughput techniques, both for bio-interaction studies by immunoassays and for mass identification of biomolecules by the use of mass spectrometry (MS). Considering that it only takes microseconds for MS spectra generation, sample preparation from biological materials is currently the rate limiting step when running large sample numbers. The use of micromachined tools for sample handling has a number of advantages such as low manufacturing cost in batch processing, high mechanical strength and reproducible structures when fabricating well-defined and small flow channels, minute orifices, and thin membranes. In this respect monocrystalline silicon is an excellent material with mechanical properties in many aspects comparable to stainless steel. This combined with its chemical robustness and the amount of known surface modifications available, makes silicon a useful construction material for chemical microsystems. Miniaturization provides increased reaction kinetics in low volumes and the possibility to rapidly perform automated high precision sample-handling procedures at a high speed in micro/nanoliter systems. Since sample amounts in life science research fields are often very small and precious, it is desirable that every analysis consumes as little sample as possible while providing the desired information. This makes the combination of silicon micromachined analytical tools and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) ideal for rapid automated analysis of proteins and peptides.