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.

Cotesting for Human Immunodeficiency Virus and Sexually Transmitted Infections in the Emergency Department.

BACKGROUND: The Centers for Disease Control and Prevention (CDC) and US Preventive Services Task Force (USPSTF) guidelines recommend screening for human immunodeficiency virus (HIV) in patients aged 15 to 65 years, as well as those at increased risk. Patients screened in the emergency department (ED) for gonorrhea (GC) and/or chlamydia represent an increased-risk population. Our aim was to assess compliance with CDC and USPSTF guidelines for HIV testing in a national sample of EDs. METHODS: We examined data from the 2010 to 2018 Nationwide Emergency Department Sample, which can be used to create national estimates of ED care to query tests for GC, chlamydia, HIV, and syphilis testing. Weighted proportions and 95% confidence intervals (CIs) were reported, and Rao-Scott χ 2 tests were used. RESULTS: We identified 13,443,831 (weighted n = 3,094,214) high-risk encounters in which GC/chlamydia testing was performed. HIV screening was performed in 3.9% (95% CI, 3.4-4.3) of such visits, and syphilis testing was performed in 2.9% (95% CI, 2.7-3.2). Only 1.5% of patients with increased risk encounters received both HIV and syphilis cotesting. CONCLUSIONS: Despite CDC and USPSTF recommendations for HIV and syphilis screening in patients undergoing STI evaluation, only a small proportion of patients are being tested. Further studies exploring the barriers to HIV screening in patients undergoing STI assessment in the ED may help inform future projects aimed at increasing guidance compliance.

Misleading FT4 and FT3 Due to Immunoassay Interference From Autoantibodies.

BACKGROUND: Immunoassays used to measure total and free thyroxine (T4 and FT4) and total and free triiodothyronine (T3 and FT3) can provide inaccurate results due to interference from endogenous autoantibodies. CASE REPORT: A 74-year-old female treated for hypothyroidism with levothyroxine replacement had elevated thyroid stimulating hormone (TSH), FT4, and FT3. Due to concern for hyperthyroidism, levothyroxine was discontinued and further workup was initiated. A pituitary MRI revealed a microadenoma but the alpha-subunit was normal. She was given octreotide for suspected TSH secreting pituitary adenoma without improvement in her TSH, FT4, or FT3 levels. She was referred to our clinic, where inaccurate lab values for FT4 and FT3 were suspected. RESULTS: Testing via equilibrium dialysis liquid chromatography tandem mass spectrometry (LC-MS/MS) method revealed lower levels of FT4 and FT3. Subsequent testing included heterophile blocking tube treatment, polyethylene glycol (PEG) precipitation, and anti-T3/T4 autoantibody levels. The tests revealed thyroid hormone autoantibodies (THAAs) as the cause of immunoassay interference. CONCLUSIONS: When thyroid hormones are elevated and TSH is not suppressed, confirmatory testing with another method such as equilibrium dialysis LC-MS/MS, which is not susceptible to interference from autoantibodies, should be considered.

Nonisotopic reagents for a cost-effective increase in sample throughput of targeted quantitative proteomics.

The new technology of ultrathroughput MS (uMS) transforms the intrinsic capability of analyte multiplexing in mass spectrometry (MS) to sample multiplexing. Core technological advantages of uMS rely on the decoupled use of isotopic quantitation reference and nonisotopic mass coding of samples. These advantages include: (1) high sample-throughput potential, (2) utilization of minimal amounts of expensive stable isotopes for the quantitation reference, and (3) unleashing of the open-source exploration of the chemical structure diversity of nonisotopic reagents to significantly enhance the MS detectability of analytes. A particular uMS method, ultrathroughput multiple reaction monitoring (uMRM), is reported for one-experiment quantitation of a surrogate peptide (SVILLGR) of prostate specific antigen (PSA) in multiple serum samples. Following derivatization of the pair of spiked, isotopic reference (SVILLGR*) and endogenous, native peptide in each sample, all samples were pooled for a step of simultaneous enrichment and cleanup of derivatized peptide pairs using immobilized antibody. The MS analysis of the pooled sample reported the quantity and sample origin of the surrogate peptide. Several analyses with different sample throughput were presented, with the highest being 15-in-1. Screening of nonisotopic reagents used combinatorial libraries of peptidyl compounds, and the reagent selection was based on the derivatization effectiveness and the capability of MS signal enhancement for the peptide. The precision, accuracy, and linearity of the uMRM MS technology were found to be comparable with standard isotope dilution MRM MS.

Development of structural marker peptides for cystic fibrosis transmembrane conductance regulator in cell plasma membrane by reversed-footprinting mass spectrometry.

A targeted mass spectrometry-based method is presented that adopts the fast photochemical oxidation of proteins (FPOP) for footprinting of cystic fibrosis transmembrane conductance regulator (CFTR) membrane transporter at its original plasma membrane location. Two analytical imperatives were sought: (1) overall simplification in data acquisition and analysis and (2) lower quantitation limits, which enabled direct analysis of intrinsically low-abundance transmembrane proteins. These goals were achieved by using a reversed-footprinting technique that monitored the unoxidized peptides remaining after the FPOP treatment. In searching for structurally informative peptides, a workflow was designed for accurate and precise quantitation of CFTR peptides produced from proteolytically digesting the plasma membrane subproteome of cells. This sample preparation strategy mitigated the need for challenging purification of large quantities of structurally intact CFTR. On the basis of the interrogated peptides, it was proposed a concept of the structural marker peptide that could report CFTR structure and function in cells. The reversed-footprinting mass spectrometry extends the FPOP technology to study conformation and interaction changes of low-abundance proteins directly in their endogenous cellular locations.

Targeted proteomic quantitation of the absolute expression and turnover of cystic fibrosis transmembrane conductance regulator in the apical plasma membrane.

Deficient chloride transport through cystic fibrosis (CF) transmembrane conductance regulator (CFTR) causes lethal complications in CF patients. CF is the most common autosomal recessive genetic disease, which is caused by mutations in the CFTR gene; thus, CFTR mutants can serve as primary targets for drugs to modulate and rescue the ion channel's function. The first step of drug modulation is to increase the expression of CFTR in the apical plasma membrane (PM); thus, accurate measurement of CFTR in the PM is desired. This work reports a tandem enrichment strategy to prepare PM CFTR and uses a stable isotope labeled CFTR sample as the quantitation reference to measure the absolute amount of apical PM expression of CFTR in CFBE 41o- cells. It was found that CFBE 41o- cells expressing wild-type CFTR (wtCFTR), when cultured on plates, had 2.9 ng of the protein in the apical PM per million cells; this represented 10% of the total CFTR found in the cells. When these cells were polarized on filters, the apical PM expression of CFTR increased to 14%. Turnover of CFTR in the apical PM of baby hamster kidney cells overexpressing wtCFTR (BHK-wtCFTR) was also quantified by targeted proteomics based on multiple reaction monitoring mass spectrometry; wtCFTR had a half-life of 29.0 ± 2.5 h in the apical PM. This represents the first direct measurement of CFTR turnover using stable isotopes. The absolute quantitation and turnover measurements of CFTR in the apical PM can significantly facilitate understanding the disease mechanism of CF and thus the development of new disease-modifying drugs. Absolute CFTR quantitation allows for direct result comparisons among analyses, analysts, and laboratories and will greatly amplify the overall outcome of CF research and therapy.

Peptide dimethylation: fragmentation control via distancing the dimethylamino group.

Direct reductive methylation of peptides is a common method for quantitative proteomics. It is an active derivatization technique; with participation of the dimethylamino group, the derivatized peptides preferentially release intense a1 ions. The advantageous generation of a1 ions for quantitative proteomic profiling, however, is not desirable for targeted proteomic quantitation using multiple reaction monitoring mass spectrometry; this mass spectrometric method prefers the derivatizing group to stay with the intact peptide ions and multiple fragments as passive mass tags. This work investigated collisional fragmentation of peptides whose amine groups were derivatized with five linear ω-dimethylamino acids, from 2-(dimethylamino)-acetic acid to 6-(dimethylamino)-hexanoic acid. Tandem mass spectra of the derivatized tryptic peptides revealed different preferential breakdown pathways. Together with energy resolved mass spectrometry, it was found that shutting down the active participation of the terminal dimethylamino group in fragmentation of derivatized peptides is possible. However, it took a separation of five methylene groups between the terminal dimethylamino group and the amide formed upon peptide derivatization. For the first time, the gas-phase fragmentation of peptides derivatized with linear ω-dimethylamino acids of systematically increasing alkyl chain lengths is reported.