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Background Subclinical inflammation was shown to play a role in the context of cardiovascular disorder processes. American College of Cardiology/American Heart Association guidelines on cardiovascular risk assessment in specific clinical contexts recommend the use of C-reactive protein (CRP) measurement with high sensitive (hs)-CRP assays that meet the precision requirements for values Methods A multisite comparative study between hs-CRP and a new conventional CRP assay (Tina-quant) was performed to evaluate the possibility of using regular CRP assays for cardiovascular risk assessment. Results A satisfactory concordance was observed between regular CRP assays and the hs-CRP assay. Both assays met the analytical precision requirements at the different cutpoints tested (1.00, 2.00, and 3.00 mg/L). Conclusion These results suggest that this new regular CRP assay can be used for cardiovascular risk assessment, which is expected to provide substantial operational and financial advantages when compared with hs-CRP assays.

Background Biological diagnosis of hemoglobin disorders is a complex process relying on the combination of several analytical techniques to identify Hb variants in a particular sample. Currently, hematology laboratories usually use high-performance liquid chromatography (HPLC), capillary electrophoresis and gel-based methods to characterize Hb variants. Co-elution and co-migration may represent major issues for precise identification of Hb variants, even for the most common ones such as Hb S and C. Methods We adapted a top-down selected reaction monitoring (SRM) electron transfer dissociation (ETD) mass spectrometry (MS) method to fit with a clinical laboratory environment. An automated analytical process with semi-automated data analysis compatible with a clinical practice was developed. A comparative study between a reference HPLC method and the MS assay was performed on 152 patient samples. Results The developed workflow allowed to identify with high specificity and selectivity the most common Hb variants (Hb S and Hb C). Concordance of the MS-based approach with HPLC was 71/71 (100%) for Hb S and 11/11 (100%) for Hb C. Conclusions This top-down SRM ETD method can be used in a clinical environment to detect Hb S and Hb C.

The application of mass spectrometry (MS)-based methods for the analysis of proteins in clinical chemistry has been a source of inspiration for many years (1, 2). Today, however, despite substantial advances in methodology and instrumentation, protein MS is still far from being the standard in clinical chemistry laboratories. MS's ability to generate complex protein or peptide profiles that can be compared among samples was initially recognized as a powerful approach for characterizing pathophysiological states and therefore a promising tool for disease diagnosis and prognosis. This strategy works well with low-complexity proteomes of simple organisms and has allowed MALDI-TOF MS protein profiling to revolutionize the identification of bacteria and fungi in clinical microbiology laboratories (3). However, when applied to more complex samples, such as human blood or urine, the lack of reproducibility of MS proteomic profiling methods and the difficulty in extracting useful information from a vast background of nonmeaningful data were soon considered insurmountable obstacles to clinical application. Because of these limitations, the prevailing view became that, to integrate clinical laboratories, MS protein assays should rely on simpler analytical work flows and straightforward data analysis processes. In other words, rather than comparing long protein lists as done in discovery proteomics, clinical proteomics should focus on the quantification of specific protein biomarkers of relevant clinical interest, taking advantage of the higher analytical performance MS can offer over traditional immunoassays (4, 5). Such targeted MS assays typically combine a protein digestion step, the fractionation of the resulting peptides by liquid chromatography, and the quantification of signature peptides by MS. An immuno-enrichment step of proteins or peptides can be added to increase …

Characterization of protein structure modifications is an important field in mass spectrometry (MS)-based proteomics. Here, we describe a process to quickly and reliably identify a mass change in a targeted protein sequence by top-down mass spectrometry (TD MS) using electron transfer dissociation (ETD). The step-by-step procedure describes how to develop a TD MS method for data acquisition as well as the data analysis process. The described TD MS workflow utilizes diagnostic ions to characterize an unknown sample in a few hours.

Precise and accurate quantification of proteins is essential in clinical laboratories. Here, we present a mass spectrometry (MS)-based method for the quantification of intact proteins in an ion trap mass spectrometer. The developed method is based on the isolation and detection of precursor ions for the quantification of the corresponding signals. The method was applied for the quantification of hemoglobin (Hb) A2, a marker used for the diagnosis of a β-thalassemia trait. The α and δ globin chains, corresponding to total Hb and HbA2, respectively, were isolated in the ion trap at specific charge states and ejected without activation. Areas of the corresponding isolated precursor ions were used to calculate the δ to α ratio. Three series of quantifications were performed on 7 different days. The standard curve fitted linearly (R(2) = 0.9982) and allowed quantification of HbA2 over a concentration range from 3% to 18% of total Hb. Analytical imprecision ranged from 3.5% to 5.3%, which is enough to determine if the HbA2 level is below 3.5% or above 3.7%. In conclusion, our method reaches precision requirements that would be acceptable for the quantitative measurement of diagnostic proteins, such as HbA2, in clinical laboratories.

A mass spectrometry-based assay combining the specificity of selected reaction monitoring and the protein ion activation capabilities of electron transfer dissociation was developed and employed for the rapid identification of hemoglobin variants from whole blood without previous proteolytic cleavage. The analysis was performed in a robust ion trap mass spectrometer operating at nominal mass accuracy and resolution. Subtle differences in globin sequences, resulting with mass shifts of about one Da, can be unambiguously identified. These results suggest that mass spectrometry analysis of entire proteins using electron transfer dissociation can be employed on clinical samples in a workflow compatible with diagnostic applications.