Multicenter Study Demonstrates Standardization Requirements for Mold Identification by MALDI-TOF MS.

OBJECTIVES: Rapid and accurate mold identification is critical for guiding therapy for mold infections. MALDI-TOF MS has been widely adopted for bacterial and yeast identification; however, few clinical laboratories have applied this technology for routine mold identification due to limited database availability and lack of standardized processes. Here, we evaluated the versatility of the NIH Mold Database in a multicenter evaluation. METHODS: The NIH Mold Database was evaluated by eight US academic centers using a solid media extraction method and a challenge set of 80 clinical mold isolates. Multiple instrument parameters important for spectra optimization were evaluated, leading to the development of two specialized acquisition programs (NIH method and the Alternate-B method). RESULTS: A wide range in performance (33-77%) was initially observed across the eight centers when routine spectral acquisition parameters were applied. Use of the NIH or the Alternate-B specialized acquisition programs, which are different than those used routinely for bacterial and yeast spectral acquisition (MBT_AutoX), in combination with optimized instrument maintenance, improved performance, illustrating that acquisition parameters may be one of the key limiting variable in achieving successful performance. CONCLUSION: Successful mold identification using the NIH Database for MALDI-TOF MS on Biotyper systems was demonstrated across multiple institutions for the first time following identification of critical program parameters combined with instrument optimization. This significantly advances our potential to implement MALDI-TOF MS for mold identification across many institutions. Because instrument variability is inevitable, development of an instrument performance standard specific for mold spectral acquisition is suggested to improve reproducibility across instruments.

Immunomics in Skin Cancer - Improvement in Diagnosis, Prognosis and Therapy Monitoring.

This review will focus on the elements of the skin's immune system, immune cells and/or non-immune cells that support immune mechanisms, molecules with immune origin and/or immune functions that are involved in skin carcinogenesis. All these immune elements are compulsory in the development of skin tumors and/or sustainability of the neoplastic process. In this light, recent data gathered in this review will acknowledge all immune elements that contribute to skin tumorigenesis; moreover, they can serve as immune biomarkers. These immune markers can contribute to the diagnostic improvement, prognosis forecast, therapy monitoring, and even personalized therapeutical approach in skin cancer. Immune processes that sustain tumorigenesis in non-melanoma and melanoma skin cancers are described in the framework of recent data.

Purification and identification of candidate biomarkers discovered using SELDI-TOF MS.

Purification and identification of candidate biomarkers is a critical step in the biomarker development process, since it provides insight into the disease biology and facilitates the development of analyte-specific assays. Top-down biomarker discovery workflows like SELDI-TOF MS yield candidate markers that are identified based on native mass. Positive identification of these candidate biomarkers requires further enrichment and/or purification. While purification methods must be optimized for each protein target, there are two general workflows. Native peptides under approximately 4 kDa can be subjected to direct sequence analysis using a tandem mass spectrometer whereas proteins over approximately 4 kDa usually require proteolytic digestion prior to MS/MS analysis. In both cases, partial purification is usually necessary to enrich the candidate biomarker relative to other proteins in a complex biological mixture. This chapter provides detailed protocols for protein purification (including anion exchange, metal affinity, and reverse phase chromatography as well as SDS-PAGE) and identification (including protein processing, digestion, and database searching).

Profiling of urine using ProteinChip® technology.

Urine is an extremely valuable sample type for biomarker discovery due to the non-invasive collection and the relatively low protein content, which makes detection of perturbations associated with disease easier. SELDI-TOF analysis is ideally suited for analysis of urine since the chromatographic capture mechanism can tolerate salt and urea in the urine sample that would otherwise need to be removed prior to mass spectrometric analysis. While neat urine can be analyzed directly on ProteinChip arrays, urine can also benefit from an enrichment step, which has been shown to increase the number of proteins detected more than twofold. Because urine volume and contents can vary substantially between individuals and within individuals over time, sample collection and storage should be carefully controlled to assure reproducible and clinically relevant results.

The Lucid Proteomics System for top-down biomarker research.

Advances have been made in recent years for both "top-down" and "bottom-up" profiling approaches to biomarker discovery. Top-down protein profiling via SELDI-TOF mass spectrometry has been used by researchers in many fields of study to discover native protein biomarker candidates from a variety of sample types, but has been limited without a means for straightforward identification of these candidates. Bio-Rad has recently partnered with Bruker Daltonics to create the Lucid Proteomics System, a complete SELDI-based research workflow--system qualification, biomarker discovery, data analysis, and biomarker purification/identification--using Bruker's flex series of TOF and TOF/TOF mass spectrometers, which have long provided consistent performance and high value data for MALDI applications. This collaboration enables both top-down and bottom-up proteomics approaches on a single high performance MALDI-TOF MS platform for maximum coverage of the proteome--allowing greater flexibility with experimental design and accelerating biomarker research programmes.

Role of trehalose and heat in the structure of the C-terminal activation domain of the heat shock transcription factor.

The heat shock transcription factor (HSF) is the primary transcriptional regulator of the heat shock response in eukaryotes. Saccharomyces cerevisiae HSF1 has two functional transcriptional activation domains, located N- and C-terminal to the central core of the protein. These activation domains have a low level of transcriptional activity prior to stress, but they acquire a high level of transcriptional activity in response to stresses such as heat. Previous studies on the N-terminal activation domain have shown that it can be completely disordered. In contrast, we show that the C-terminal activation domain of S. cerevisiae HSF1 does contain a certain amount of secondary structure as measured by circular dichroism (CD) and protease resistance. The alpha-helical content of the domain can be increased by the addition of the disaccharide trehalose but not by sucrose. Trehalose, but not sucrose, causes a blue shift in the fluorescence emission spectra, which is suggestive of an increase in tertiary structure. Trehalose, which is known to be a chemical chaperone, also increases proteases' resistance and promotes heat-induced increases in alpha-helicity. The latter is particularly intriguing because of the physiological role of trehalose in yeast. Trehalose levels are increased dramatically after heat shock, and this is thought to protect protein structure prior to the increase of heat shock protein levels. Our results suggest that the dramatic changes in S. cerevisiae HSF1 transcriptional activity in response to stress might be linked to the combined effects of trehalose and elevated temperatures in modifying the overall structure of HSF1's C-terminal activation domain.