Mark S. Lowenthal

Biomarker amplification by serum carrier protein binding.

Mass spectroscopic analysis of the low molecular mass (LMM) range of the serum/plasma proteome is a rapidly emerging frontier for biomarker discovery. This study examined the proportion of LMM biomarkers, which are bound to circulating carrier proteins. Mass spectroscopic analysis of human serum following molecular mass fractionation, demonstrated that the majority of LMM biomarkers exist bound to carrier proteins. Moreover, the pattern of LMM biomarkers bound specifically to albumin is distinct from those bound to non-albumin carriers. Prominent SELDI-TOF ionic species (m/z 6631.7043) identified to correlate with the presence of ovarian cancer were amplified by albumin capture. Several insights emerged: a) Accumulation of LMM biomarkers on circulating carrier proteins greatly amplifies the total serum/plasma concentration of the measurable biomarker, b) The total serum/plasma biomarker concentration is largely determined by the carrier protein clearance rate, not the unbound biomarker clearance rate itself, and c) Examination of the LMM species bound to a specific carrier protein may contain important diagnostic information. These findings shift the focus of biomarker detection to the carrier protein and its biomarker content.

Discovering Clinical Biomarkers of Ionizing Radiation Exposure with Serum Proteomic Analysis

In this study, we sought to explore the merit of proteomic profiling strategies in patients with cancer before and during radiotherapy in an effort to discover clinical biomarkers of radiation exposure. Patients with a diagnosis of cancer provided informed consent for enrollment on a study permitting the collection of serum immediately before and during a course of radiation therapy. High-resolution surface-enhanced laser desorption and ionization-time of flight (SELDI-TOF) mass spectrometry (MS) was used to generate high-throughput proteomic profiles of unfractionated serum samples using an immobilized metal ion-affinity chromatography nickel-affinity chip surface. Resultant proteomic profiles were analyzed for unique biomarker signatures using supervised classification techniques. MS-based protein identification was then done on pooled sera in an effort to begin to identify specific protein fragments that are altered with radiation exposure. Sixty-eight patients with a wide range of diagnoses and radiation treatment plans provided serum samples both before and during ionizing radiation exposure. Computer-based analyses of the SELDI protein spectra could distinguish unexposed from radiation-exposed patient samples with 91% to 100% sensitivity and 97% to 100% specificity using various classifier models. The method also showed an ability to distinguish high from low dose-volume levels of exposure with a sensitivity of 83% to 100% and specificity of 91% to 100%. Using direct identity techniques of albumin-bound peptides, known to underpin the SELDI-TOF fingerprints, 23 protein fragments/peptides were uniquely detected in the radiation exposure group, including an interleukin-6 precursor protein. The composition of proteins in serum seems to change with ionizing radiation exposure. Proteomic analysis for the discovery of clinical biomarkers of radiation exposure warrants further study.

CSF proteome: a protein repository for potential biomarker identification

Proteomic analysis is not limited to the analysis of serum or tissues. Synovial, peritoneal, pericardial and cerebrospinal fluid represent unique proteomes for disease diagnosis and prognosis. In particular, cerebrospinal fluid serves as a rich source of putative biomarkers that are not solely limited to neurologic disorders. Peptides, proteolytic fragments and antibodies are capable of crossing the blood–brain barrier, thus providing a repository of pathologic information. Proteomic technologies such as immunoblotting, isoelectric focusing, 2D gel electrophoresis and mass spectrometry have proven useful for deciphering this unique proteome. Cerebrospinal fluid proteins are generally less abundant than their corresponding serum counterparts, necessitating the development and use of sensitive analytical techniques. This review highlights some of the promising areas of cerebrospinal fluid proteomic research and their clinical applications.

Development of a Standard Reference Material for Metabolomics Research

The National Institute of Standards and Technology (NIST), in collaboration with the National Institutes of Health (NIH), has developed a Standard Reference Material (SRM) to support technology development in metabolomics research. SRM 1950 Metabolites in Human Plasma is intended to have metabolite concentrations that are representative of those found in adult human plasma. The plasma used in the preparation of SRM 1950 was collected from both male and female donors, and donor ethnicity targets were selected based upon the ethnic makeup of the U.S. population. Metabolomics research is diverse in terms of both instrumentation and scientific goals. This SRM was designed to apply broadly to the field, not toward specific applications. Therefore, concentrations of approximately 100 analytes, including amino acids, fatty acids, trace elements, vitamins, hormones, selenoproteins, clinical markers, and perfluorinated compounds (PFCs), were determined. Value assignment measurements were performed by NIST and the Centers for Disease Control and Prevention (CDC). SRM 1950 is the first reference material developed specifically for metabolomics research.

Identification of Novel N-Glycosylation Sites at Noncanonical Protein Consensus Motifs

N-glycosylation of proteins is well known to occur at asparagine residues that fall within the canonical consensus sequence N-X-S/T but has also been identified at a small number of asparagine residues within N-X-C motifs, including the N491 residue of human serotransferrin. Here we report novel glycosylation sites within noncanonical consensus motifs, in the conformation N-X-C, based on mass spectrometry analysis of partially deglycosylated glycopeptide targets. Alpha-1-acid glycoprotein (A1AG) and serotransferrin (Tf) were observed for the first time to be N-glycosylated on asparagine residues within a total of six unique noncanonical motifs. N-glycosylation was initially predicted in silico based on the evolutionary conservation of the N-X-C motif among related mammalian species and demonstrated experimentally in A1AG from porcine, canine, and feline sources and in human serotransferrin. High-resolution liquid chromatography-tandem mass spectrometry was employed to collect fragmentation data of predicted GlcNAcylated peptides and to assign modification sites within N-X-C motifs. A combination of targeted analytical techniques that includes complementary mass spectrometry platforms, enzymatic digestions, and partial-deglycosylation procedures was developed to confirm the novel observations. Additionally, we found that A1AG in porcine and canine sources is highly N-glycosylated at a noncanonical motif (N-Q-C) based on semiquantitative multiple reaction monitoring analysis-the first report of an N-X-C motif exhibiting substantial N-glycosylation. Although reports of N-X-C motif N-glycosylation are relatively uncommon in the literature, this work adds to a growing list of glycoproteins reported with glycosylation at various forms of noncanonical motifs.