High Throughput Peptide Mass Fingerprinting and Protein Macroarray Analysis Using Chemical Printing Strategies

107

We describe a 96-well plate compatible membrane-based proteomic sample processing method, which enables the complete processing of 96 samples (or multiples thereof) within a single workday. This method uses a large-pore hydrophobic PVDF membrane that efficiently adsorbs proteins, resulting in fast liquid transfer through the membrane and significantly reduced sample processing times. Low liquid transfer speeds have prevented the useful 96-well plate implementation of FASP as a widely used membrane-based proteomic sample processing method. We validated our approach on whole-cell lysate and urine and cerebrospinal fluid as clinically relevant body fluids. Without compromising peptide and protein identification, our method uses a vacuum manifold and circumvents the need for digest desalting, making our processing method compatible with standard liquid handling robots. Mass spectrometry (MS)-based proteomics is moving increasingly into the translational and clinical research arena, where robust and efficient sample processing is of particular importance. The conventional sample processing methods in proteomics, namely SDS-PAGE, or in-solution-based sample processing, are slow and laborious and thus do not easily provide the reproducibility and throughput to meet current demands. A paradigm shift was the introduction of a filteraided sample processing method (FASP), which is initially described by Manza et al.(1) and then fully realized in practice by Wisniewski et al. (2). These filter-aided methods make use of ultrafiltration membranes with molecular weight cut offs (MWCO) in the 10 to 30 kDa range to efficiently remove small molecules and salts and to capture denatured proteins on a cellulose filter even if the molecular weight of the protein is much smaller than the nominal MWCO of the ultrafiltration membrane. Thus, the denaturation step is crucial to ensure that proteins much smaller than the nominal MWCO are efficiently retained by, e.g. a 10 kDa MWCO filter.In translational and clinical proteomics, which normally include large cohorts, the multititer-well plate is the preferred format for sample processing and storage. Although the application of FASP in the 96-well plate format has been described (3, 4), the major limitation of FASP in the 96-well plate is the much slower speed at which the 96-well plates have to be centrifuged: while a single ultrafiltration unit withstands up to 14,000 ϫ g, the 96-well plate format can only be centrifuged at g-forces of up to 2,200 ϫ g. This significantly lower g-force for 96-well plates results in a slow liquid transfer, which in turn considerably prolongs the required centrifugation times to hours instead of tens of minutes for the three to four necessary centrifugation steps (i) for the initial loading, reduction and alkylation, (ii) for the different washing steps, and (iii) for the elution (3).Independent of the format FASP is performed in, the conventional FASP also requires relative large volumes of high salt concentration for efficient elution of the tryptic peptid...

Section: Resultsmentioning

confidence: 99%

MStern Blotting–High Throughput Polyvinylidene Fluoride (PVDF) Membrane-Based Proteomic Sample Preparation for 96-Well Plates

Berger

Ahmed

Muntel

et al. 2015

107

“…Captured antigens are separated by 2-DE to resolve protein isoforms for multiple analyses of each captured protein, including MS-based identification and characterization. Furthermore we demonstrate how gel-extracted protein aliquots can be used for instant validation of protein immunogenicity across multiple clinical samples using a chemical inkjet printer, ChIP TM (14). We demonstrate application of an immunoproteomic strategy to identify a panel of antigenic biomarkers for pulmonary exacerbation in cystic fibrosis (CF).…”

mentioning

confidence: 99%

An Immunoproteomic Approach for Identification of Clinical Biomarkers for Monitoring Disease

Pedersen

Sloane

Prasad

et al. 2005

Self Cite

Circulating antibodies can be used to probe protein arrays of body fluids, prepared by two-dimensional gel electrophoresis, for antigenic biomarker detection. However, detected proteins, particularly low abundance antigens, often remain unidentifiable due to proteome complexity and limiting sample amounts. Using a novel enrichment approach exploiting patient antibodies for isolation of antigenic biomarkers, we demonstrate how immunoproteomic strategies can accelerate biomarker discovery. Application of this approach as a means of identifying biomarkers was demonstrated for cystic fibrosis (CF) lung disease by isolation and identification of inflammatoryassociated autoantigens, including myeloperoxidase and calgranulin B from sputum of subjects with CF. The approach was also exploited for isolation of proteins expressed by the Pseudomonas aeruginosa strain PA01. Capture of PA01 antigens using circulating antibodies from CF subjects implicated in vivo expression of Pseudomonas proteins. All CF subjects screened, but not controls, were immunoreactive against immunocaptured Pseudomonas proteins, representing stress (GroES and ferric iron-binding protein HitA), immunosuppressive (thioredoxin), and alginate synthetase pathway (nucleosidediphosphate kinase) proteins, implicating their clinical relevance as biomarkers of infection.

“…A number of groups, including our own, are exploring robotic devices as tools for increasing the reproducibility of submicroliter volumes of matrix solution deposited onto tissue sections (21)(22)(23)(24)(25). Microspots of matrix with diameters on the order of 200 -300 m can be generated with greater speed and precision than manual methods, allowing arrays of hundreds of matrix microspots to be deposited in a matter of minutes.…”

mentioning

confidence: 99%

A Novel Histology-directed Strategy for MALDI-MS Tissue Profiling That Improves Throughput and Cellular Specificity in Human Breast Cancer

Cornett

Mobley

Dias

et al. 2006

175

149

We describe a novel tissue profiling strategy that improves the cellular specificity and analysis throughput of protein profiles obtained by direct MALDI analysis. The new approach integrates the cellular specificity of histology, the accuracy and reproducibility of robotic liquid dispensing, and the speed and objectivity of automated spectra acquisition. Traditional methodologies for preparing and analyzing tissue samples rely heavily on manual procedures, which for various reasons discussed, restrict cellular specificity and sample throughput. Here, a robotic spotter deposits micron-sized droplets of matrix precisely onto foci of normal mammary epithelium, ductal carcinoma in situ, invasive mammary cancer, and peritumoral stroma selected by a pathologist from high resolution histological images of sectioned human breast cancer samples. The location of each matrix spot was then determined and uploaded into the instrument to facilitate automated profile acquisition by MALDI-TOF. In the example shown, the different lesions were clearly differentiated using mass profiling. Further, the workflow permits a visual projection of any information produced from the profile analyses directly on the histological image for a unique combination of proteomic and histological assessment of sample regions. The higher performance characteristics offered by the new workflow promises to be a significant advancement toward the next generation of tissue profiling studies. Molecular & Cellular Proteomics 5: 1975-1983, 2006.