Formalin Fixation Followed by Paraffin Embedding Allows Long-Term Storage of Proteins for Liquid Chromatography-Tandem Mass Spectrometry Analysis.

In an anatomical pathology laboratory, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is used to characterize amyloid deposits identified in formalin-fixed paraffin-embedded tissue (FFPET). However, the development of additional tests is partially limited by the lack of information the passage of time has on the proteins in FFPET. To investigate the reliability of LC-MS/MS in the analysis of old FFPET specimens, 1 bone marrow aspirate clot was analyzed by LC-MS/MS yearly from 2014 to 2018, in 3 consecutive months. Peptide-spectrum match, number of peptides identified, and percentage of the proteins covered were the parameters collected for the hemoglobin subunits alpha (HbA), beta (HbB), delta (HbD), and gamma (HbG). These proteins are constant components of the peripheral blood and are present in high and low abundance, allowing the monitorization of the performance of the test across varying protein concentrations. The hemoglobin subunits were stable over the years studied; 71% to 74% of HbA, 77% to 80% of HbB, 69% to 77% of HbD, and 57% to 63% of HbG were covered, with no statistical difference between 2014 and 2018. The number of peptides identified was also constant, 11 to 13 for HbA, 13 to 15 for HbB, 11 to 14 for HbD, and 7 to 9 for HbG. Peptide spectrum match was only slightly more variable: 209 to 327 for HbA, 569 to 1052 for HbB, 286 to 533 HbD, and 142 to 292 for HbG. In conclusion, high abundance hemoglobins, HbA and HbB, and relatively low abundance ones, HbD and HbG, are preserved in FFPET and confidently identified by LC-MS/MS for at least 5 years.

A comprehensive and comparative proteomic analysis of horse serum proteins in colitis.

BACKGROUND: Equine colitis is a diarrhoeal disease caused by inflammation of the large bowel and can potentially be life-threatening due to its rapid progression. Pathogenesis is multifactorial and pathophysiology is highly complicated, therefore, reliable diagnostic biomarkers are needed in the veterinary field. OBJECTIVE: Serum is one of the most commonly used diagnostic tools in equine clinical investigation. To discover diagnostic or prognostic protein markers for colitis in horse serum, comprehensive and comparative proteomic analysis was conducted using liquid chromatography-tandem mass spectrometry (LC-MS/MS). STUDY DESIGN: Case-control study. METHODS: Serum samples were collected from 36 healthy Thoroughbreds and 12 Thoroughbreds with colitis. Serum from each horse suffering from colitis was collected daily until death or recovery. Collected sera were digested with trypsin. Peptides obtained from serum proteins were measured by Q-Exactive HF Orbitrap mass spectrometer. The identification and quantification of peptides were performed using Proteome Discoverer version 2.2. RESULTS: On day 1 of treatment, eight proteins in the colitis group were upregulated (P < .05, more than a twofold change) compared with the healthy group. Among the eight proteins, biliverdin reductase B was significantly upregulated (P < .05) in the non-survivor group (n = 5) compared with the survivor group (n = 7). On the last day of the treatment, haemoglobin subunit alpha, clusterin, glyceraldehyde-3-phosphate dehydrogenase, lipopolysaccharide-binding protein, and biliverdin reductase B showed significant increases (P < .05) in the non-survivor group. MAIN LIMITATIONS: The number of the identified proteins is limited due to the existence of abundant proteins. CONCLUSIONS: Measuring the changes of these proteins together may enable a potential prognosis or early diagnosis of horses suffering from colitis.

Evaluating the effect of body fluid mixture on the relative expression ratio of blood-specific RNA markers.

The estimation of the time elapsed since a biological stain was deposited at a crime scene can provide crucial information to a forensic investigation, indicating either when a crime was committed, or whether the biological evidence was deposited at the time of a known crime event. This would enable the investigators to limit the number of suspects and to assess alibis. The relative expression ratios (RERs) of body fluid-specific RNA markers are promising molecular tools for indicating the age of biological stains. However, the nature of some forensic samples found at crime scenes could be challenging, as they frequently occur in a mixture of different body fluid types. The research presented here has utilised reverse transcription quantitative PCR (RT-qPCR) to explore the impact of bloodstains being present in mixtures with other body fluids (saliva or semen) on the resulting RERs of blood-specific markers. The expression level of three blood-specific markers (HBA, HBB and miR16) along with two reference genes (18S and U6) were analysed across multiple ageing time points in pure and mixed bloodstains. For some markers, no significant differences were found when comparing RERs in pure and mixed bloodstains, however some RERs were altered in mixed stains. This indicates that the presence of body fluid mixtures may have a significant effect on the RERs of some blood-specific markers. This should therefore be considered when selecting markers for estimating the age of stains, particularly when multiple body fluids are thought to be present.

Quadrupole-Time-of-Flight Mass Spectrometric Identification of Hemoglobin Subunits α, ß, γ and δ in Unknown Peaks of High Performance Liquid Chromatography of Hemoglobin in ß-Thalassemias.

This is the first report of quadrupole time-of-flight (Q-TOF) mass spectrometric identification of the hemoglobin (Hb) subunits, α, ß, δ and γ peptides, derived from enzymatic-digestion of proteins in the early unknown peaks of the cation exchange chromatography of Hb. The objectives were to identify the unknown high performance liquid chromatography (HPLC) peaks in healthy subjects and in patients with ß-thalassemia (ß-thal). The results demonstrate the existence of pools of free globin chains in red blood cells (RBCs). The α-, ß-, δ- and γ-globin peptides were identified in the unknown HPLC peaks. The quantification and role of the free globin pool in patients with ß-thal requires further investigation. Identification of all types of Hb subunits in the retention time (RT) before 1 min. suggests that altered Hbs is the nature of these fast-eluting peaks. Relevancy of thalassemias to the protein-aggregation disorders will require review of the role of free globin in the pathology of the disease.

Analysis and Quantitation of Glycated Hemoglobin by Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry.

Measurement of glycated hemoglobin is widely used for the diagnosis and monitoring of diabetes mellitus. Matrix assisted laser desorption/ionization (MALDI) time of flight (TOF) mass spectrometry (MS) analysis of patient samples is used to demonstrate a method for quantitation of total glycation on the ß-subunit of hemoglobin. The approach is accurate and calibrated with commercially available reference materials. Measurements were linear (R(2) > 0.99) across the clinically relevant range of 4% to 20% glycation with coefficients of variation of ≤ 2.5%. Additional and independent measurements of glycation of the α-subunit of hemoglobin are used to validate ß-subunit glycation measurements and distinguish hemoglobin variants. Results obtained by MALDI-TOF MS were compared with those obtained in a clinical laboratory using validated HPLC methodology. MALDI-TOF MS sample preparation was minimal and analysis times were rapid making the method an attractive alternative to methodologies currently in practice.

FIT testing: an overview.

Colorectal cancer (CRC) is a common, but preventable, disease and is the second most common cause of cancer-related deaths in the U.S. CRC screening has proven effective at reducing both the incidence and mortality of this disease, using any of a number of screening tests available. The test options range from the least invasive and least expensive to more invasive and costly options. Fecal occult blood testing is the oldest, least expensive, and least invasive of these options and has evolved from the poorly sensitive standard guaiac test to the newer and diagnostically superior fecal immunochemical test (FIT) for hemoglobin. This article explores the evolutionary history of fecal occult blood testing, examines test performance characteristics among different FOBTs, and evaluates the role of the FIT in programmatic CRC screening.

Mass spectrometry-based proteomics as a tool to identify biological matrices in forensic science.

In forensic casework analysis, identification of the biological matrix and the species of a forensic trace, preferably without loss of DNA, is of major importance. The biological matrices that can be encountered in a forensic context are blood (human or non-human), saliva, semen, vaginal fluid, and to a lesser extent nasal secretions, feces, and urine. All these matrices were applied on swabs and digested with trypsin in order to obtain peptides. These peptides were injected on a mass spectrometer (ESI Q-TOF) resulting in the detection of several biomarkers that were used to build a decision tree for matrix identification. Saliva and blood were characterized by the presence of alpha-amylase 1 and hemoglobin, respectively. In vaginal fluid, cornulin, cornifin, and/or involucrin were found as biomarkers while semenogelin, prostate-specific antigen, and/or acid phosphatase were characteristic proteins for semen. Uromodulin or AMBP protein imply the presence of urine, while plunc protein is present in nasal secretions. Feces could be determined by the presence of immunoglobulins without hemoglobin. The biomarkers for the most frequently encountered biological matrices (saliva, blood, vaginal fluid, and semen) were validated in blind experiments and on real forensic samples. Additionally, by means of this proteomic approach, species identification was possible. This approach has the advantage that the analysis is performed on the first "washing" step of the chelex DNA extraction, a solution which is normally discarded, and that one single test is sufficient to determine the identity and the species of the biological matrix, while the conventional methods require cascade testing. This technique can be considered as a useful additional tool for biological matrix identification in forensic science and holds the promise of further automation.

Rapid determination of human globin chains using reversed-phase high-performance liquid chromatography.

Reversed-phase high-performance liquid chromatography (RP-HPLC) of human globin chains is an important tool for detecting thalassemias and hemoglobin variants. The challenges of this method that limit its clinical application are a long analytical time and complex sample preparation. The aim of this study was to establish a simple, rapid and high-resolution RP-HPLC method for the separation of globin chains in human blood. Red blood cells from newborns and adults were diluted in deionized water and injected directly onto a micro-jupiter C18 reversed-phase column (250 mm × 4.6 mm) with UV detection at 280 nm. Under the conditions of varying pH or the HPLC gradient, the globin chains (pre-ß, ß, δ, α, (G)γ and (A)γ) were denatured and separated from the heme groups in 12 min with a retention time coefficient of variation (CV) ranging from 0.11 to 1.29% and a peak area CV between 0.32% and 4.86%. Significant differences (P<0.05) among three groups (normal, Hb H and ß thalassemia) were found in the area ratio of α/pre-ß+ß applying the rapid elution procedure, while P≥0.05 was obtained between the normal and α thalassemia silent/trait group. Based on the ANOVA results, receiver operating characteristic (ROC) curve analysis of the δ/ß and α/pre-ß+ß area ratios showed a sensitivity of 100.0%, and a specificity of 100.0% for indicating ß thalassemia carriers, and a sensitivity of 96.6% and a specificity of 89.6% for the prediction of hemoglobin H (Hb H) disease. The proposed cut-off was 0.026 of δ/ß for ß thalassemia carriers and 0.626 of α/pre-ß+ß for Hb H disease. In addition, abnormal hemoglobin hemoglobin E (Hb E) and Hb Westmead (Hb WS) were successfully identified using this RP-HPLC method. Our experience in developing this RP-HPLC method for the rapid separation of human globin chains could be of use for similar work.

Serum proteomic profiling of obese patients: correlation with liver pathology and evolution after bariatric surgery.

OBJECTIVE: Chronic liver diseases, including cirrhosis, may develop in obese patients. Steatosis and non-alcoholic steatohepatitis (NASH) are risk factors for progression to fibrosis. To date, diagnosis of steatosis and NASH relies on liver biopsy. The aim of the study was to identify serum markers of steatosis and NASH in obese patients using SELDI-TOF ProteinChip. PATIENTS: Eighty obese non-alcoholic patient candidates for bariatric surgery and devoid of hepatitis B and C infection were selected. Serum samples were collected before surgery and at 6 months after surgery for 33 of these patients. Wedge liver biopsy was performed at the time of bariatric surgery. Twenty-four serum samples from healthy blood donors served as controls. The protein profiles of each serum were assessed using SELDI-TOF ProteinChip technology and were compared according to liver histological lesions. RESULTS: Twenty-four obese patients (30%) had non-significant liver lesions, 32 (40%) had significant steatosis and 24 (30%) had NASH. Comparison of serum protein profiles according to liver lesions identified three peaks (CM10-7558.4, CM10-7924.2 and Q10-7926.9) the intensity of which significantly increased according to the severity of the liver lesions (steatosis and NASH) and returned to normal after bariatric surgery. None was correlated with either liver function tests or metabolic parameters. Identification using immunoSELDI assay characterised these peaks as the double charged ions of alpha- and beta-haemoglobin subunits. CONCLUSION: The differential proteomic method demonstrated changes in serum protein profiles in obese patients according to severity of liver lesions. Free haemoglobin subunits may serve as a serum biomarker of the severity of liver damages.