Sample Quality Assessment
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The determination of sample quality is an important issue to ensure validity of results. Particularly in the field of multiplex assays it is a prerequisite for effective study outcomes. Sample Quality Scoring by Peptidomics is a versatile tool to qualify biological specimens. The analysis of pre-analytical biases allow for characterization of samples, detection of outliers and recognition of erroneous sample handling procedures.

Analysis of blood specimens to demonstrate the concept of Sample Quality Assessment

Pre-analytical procedures can confound the outcome of preclinical and clinical studies. In particular sample collection and specimen preparation can alter the validity of results obtained by modern multiplex assays (e.g. LC-MS). Quality assessment of collected samples is crucial to avoid systematic biases. In order to address pre-analytical biases we applied Peptidomics® to grade samples. The analysis of native peptides (Low Molecular Weight [LMW-] proteome) in biological specimens is primarily achieved by chromatographic methods coupled to multiple forms of mass spectrometry. Peptides as products of proteolytic cleavage events exhibit a close connection to protease activity. Increased or altered activity of proteases during sample collection, specimen generation, sample storage and processing are mirrored by alterations in abundance of specific peptides. To demonstrate this approach we analyze different blood specimens to elucidate biological events occurring during the pre-analytical phase by Peptidomics® and qualify the specimens by Sample Quality Assessment (SQA).

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Peptide displays from blood specimens
Peptide displays of the 6 blood specimens (EDTA plasma, citrate plasma, CTAD plasma, serum with and without clot activator and P100v1). The x-axis displays the mass-to-charge ratio, the y-axis is determined by the retention time on the RP-HPLC. The signal intensity is depicted by color saturation.


Characterization of Blood Specimens by Sample Quality Assessment

To assess the quality of a given sample an aliquot of the respective sample is analyzed via Peptidomics® to obtain peptide profiles: briefly, peptides were extracted and separated in 96 fractions by liquid chromatography. An aliquot of each fraction was subjected to MALDI mass spectrometry. Data analysis is carried out to retrieve significant mass spectrometric signals representing surrogate peptides for pre-analytical events. By analyzing the abundance of these surrogate peptides (n=77) distinct biological events (coagulation (23), kallikrein activation (18), complement activation (8) and cell lysis (25)) and their significance can be attributed to each sample to ensure comparability between samples. EDTA, Citrate and CTAD plasma exhibit a more similar pattern in comparison to Serum and P100v1. The latter specimens show signs of cell lysis, coagulation and activation of kallikrein. An in-depth analysis of EDTA, Citrate and CTAD plasma also demonstrate pronounced differences between these specimens. The main purpose of this study was to demonstrate the massive impact on generation of artificial ex-vivo generated peptides within the different specimens. The foremost application of this approach would be the characterization of sample quality inside study populations to determine possible pre-analytical biases.

Sample Quality Chart

Peptide surrogate markers were exported and analyzed according to distinct biological events.
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The left axis depicts the human blood specimen, the bottom axis the surrogate peptide (precursor abbreviation and amino acid range) and the top axis the biological event represented by groups of surrogate peptides. It is clearly visible that Serum and P100v1 exhibit significant signs of coagulation and cell lysis. Additionally the clot activator in serum samples lead to an activation of plasma kallikrein (surface activated).