Whole genome amplification of cell-free DNA enables detection of circulating tumor DNA mutations from fingerstick capillary blood.

The ability to measure mutations in plasma cell-free DNA (cfDNA) has the potential to revolutionize cancer surveillance and treatment by enabling longitudinal monitoring not possible with solid tumor biopsies. However, obtaining sufficient quantities of cfDNA remains a challenge for assay development and clinical translation; consequently, large volumes of venous blood are typically required. Here, we test proof-of-concept for using smaller volumes via fingerstick collection. Matched venous and fingerstick blood were obtained from seven patients with metastatic breast cancer. Fingerstick blood was separated at point-of-care using a novel paper-based concept to isolate plasma centrifuge-free. Patient cfDNA was then analyzed with or without a new method for whole genome amplification via rolling-circle amplification (WG-RCA). We identified somatic mutations by targeted sequencing and compared the concordance of mutation detection from venous and amplified capillary samples by droplet-digital PCR. Patient mutations were detected with 100% concordance after WG-RCA, although in some samples, allele frequencies showed greater variation likely due to differential amplification or primer inaccessibility. These pilot findings provide physiological evidence that circulating tumor DNA is accessible by fingerstick and sustains presence/absence of mutation detection after whole-genome amplification. Further refinement may enable simpler and less-invasive methods for longitudinal or theranostic surveillance of metastatic cancer.

Cerebrospinal fluid proteomic biomarkers for Alzheimer's disease.

OBJECTIVE: To find a panel of proteins in antemortem cerebrospinal fluid (CSF) that could be used to differentiate between samples from Alzheimer's disease (AD) patients and samples from healthy and neurological control subjects. METHODS: For a test cohort, antemortem CSF proteins from 34 AD and 34 non-AD patients were separated using two-dimensional gel electrophoresis. The resulting protein patterns were analyzed using the random forest multivariate statistical method. Protein spots of interest were identified using tandem time-of-flight mass spectrometry. A validation cohort consisting of CSF from 18 AD patients and 10 non-AD subjects was analyzed in a similar way. RESULTS: Using the test cohort, a panel of 23 spots was identified that could be used to differentiate AD and non-AD gels with a sensitivity of 94%, a specificity of 94%, and a predicted classification error rate of only 5.9%. These proteins are related to the transport of beta-amyloid, the inflammatory response, proteolytic inhibition, and neuronal membrane proteins. The 23 spots separately classified the validation cohort with 90% sensitivity (probable AD subjects), 83% specificity, and a predicted classification error rate of 14% in a blinded analysis. The total observed sensitivity is 93%, the total observed specificity is 90%, and the predicted classification error rate is 8.3%. INTERPRETATION: A panel of possible CSF biomarkers for AD has been identified using proteomic methods.

Proteomic analysis of cerebrospinal fluid changes related to postmortem interval.

BACKGROUND: The study of proteins with altered production in postmortem cerebrospinal fluid (CSF) compared with antemortem CSF may improve the understanding of biochemical changes that occur immediately after death. METHODS: Two CSF samples (1 antemortem and 1 postmortem) were collected from 7 patients and analyzed by 2-dimensional gel electrophoresis. An analysis was also performed to identify proteins that showed a correlation between concentration change and postmortem interval. Tandem mass spectrometry was used to identify the proteins. RESULTS: Fifty-four protein spots were identified that showed a consistent and significant change in concentration in the postmortem CSF of all 7 patients (>3.5-fold, P <0.01). The proteins in these spots derive from a variety of functional groups, including cytoskeletal proteins, enzymes involved in glycolysis, and proteins that prevent oxidative stress. Fourteen protein spots were found to have an increase in production that correlated with postmortem interval. CONCLUSIONS: Changes in protein production of postmortem vs antemortem CSF were studied. The proteins observed to change production in the postmortem CSF include several proteins previously observed as potential stroke biomarkers.

Complement protein isoforms in CSF as possible biomarkers for neurodegenerative disease.

It has been suggested that the activation of the complement system is involved in the pathogenesis of several neurodegenerative diseases including Alzheimer's disease (AD), Parkinson's disease (PD), and multiple sclerosis (MS). Here, the CSF expression levels of complement proteins C3b, C4b, factor B, and factor H were compared between normal subjects and patients diagnosed with AD, PD, MS, and neurosyphilis. The CSF proteins were initially separated using two-dimensional gel electrophoresis, which allowed the comparison of some of the individual complement isoforms. Patients with AD, PD, and MS all showed more than one complement isoform with a significant change (p < 0.05) in CSF expression level compared to normal subjects. PD patients were found to have the greatest number of significantly changed isoforms, all showing a decreased expression level in PD CSF. The complement isoforms examined were able to distinguish between some, but not all, of the diseases studied. The data suggest that when investigating a protein as a possible biomarker, it may be useful to compare individual protein isoform expression levels in addition to the more commonly measured total protein expression level.

Kinetic characterization of sequencing grade modified trypsin.

Prior to analysis by mass spectrometry, protein samples are often digested. Maximizing the peptide yield from digestion can increase the number of peptides detected and the confidence in protein identification. To determine the optimal conditions for digestion, the Michaelis-Menten kinetic parameters for Promega sequencing grade modified trypsin were measured over a range of temperatures and pHs. The results indicate that an increase in digestion temperature above 37 degrees C, the temperature traditionally used in digestion methods, could offer an increase in peptides detected.

Towards two-dimensional electrophoresis mapping of the cerebrospinal fluid proteome from a single individual.

We test the ability of state-of-the-art two-dimensional electrophoresis (2-DE) technology to enable the proteome mapping of ante mortem cerebrospinal fluid (CSF) from a single individual. Using the sensitive technologies of a fluorescent protein stain and fluorescence laser scanning of 2-DE gels, combined with matrix-assisted laser desorption/ionization-time of flight/time of flight-mass spectrometry (MALDI-TOF/TOF-MS) for protein identification, a highly detailed 2-DE map of the CSF proteome was created. The 2-DE map contains 600 identified spots representing 82 different proteins. Of the 82 proteins identified, 25 have not appeared in any previously published 2-DE map of CSF, and 11 have not been previously reported to exist in CSF. Most of the identifications originate from an ante mortem CSF sample collected from a single hydrocephalus patient. A supplemental map created from neurologically normal patients is also presented. A webpage with protein identification and scoring information from both maps is available at http://www.leelab.org/csfmap.

An introduction to mass spectrometry applications in biological research.

Mass spectrometry involves the measurement of the mass-to-charge ratio of ions. It has become an essential analytical tool in biological research and can be used to characterize a wide variety of biomolecules such as sugars, proteins, and oligonucleotides. In this review, a brief history of mass spectrometry is discussed, and the basic principles of the technology are introduced. A summary of some current applications is provided, as are examples of recently published research. The current methods used to identify, quantify, and characterize proteins and peptides are then reviewed. The range of applications of mass spectrometry is considerable and only promises to grow as the technology continues to improve.

Comparison of automated in-gel digest methods for femtomole level samples.

A comparison of automated in-gel digestion methods for low picomolar to femtomolar levels of protein is presented. Gel spots with 4 pmol to 120 fmol of protein were stained with either Coomassie colloidal blue or SYPRO Ruby and digested using an automated platform. The sequence coverages and average peak intensities obtained from a matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) analysis are compared. Results show that methods using an acetonitrile extraction or digest times greater than the standard 4 h give no significant increase in peptide sequence coverage for automated digestion of low protein level samples. It is also shown that digests from SYPRO Ruby-stained gels show a greater improvement upon ZipTip cleanup than digests from Coomassie colloidal blue-stained gels. The digests from SYPRO Ruby-stained gels are also shown to give a higher average peptide intensity if a method with minimal gel plug washing is used.