Detection and differentiation of 22 kDa and 20 kDa Growth Hormone proteoforms in human plasma by LC-MS/MS.

Human growth hormone (GH) is suspected to be widely and illegally used in sport to improve athletes' performance. For the detection of GH abuse, blood samples are screened for abnormal ratios between the 22 and 20 kDa GH proteoforms that demonstrate the administration of the synthetic hormone. Current detection methods are based on classical immunoassays as they provide sufficient sensitivity for the detection of GH proteoforms.

Integrated solid-phase extraction-capillary liquid chromatography (speLC) interfaced to ESI-MS/MS for fast characterization and quantification of protein and proteomes.

The high peptide sequencing speed provided by modern hybrid tandem mass spectrometers enables the utilization of fast liquid chromatographic (LC) separation techniques. We present a robust solid-phase extraction/capillary LC system (speLC) for 5-10 min separation of semicomplex peptide mixtures prior to ESI-MS/MS for peptide sequencing. This speLC-MS/MS system eliminates sample-to-sample carry-over by using disposable micropipette solid-phase extraction tips (StageTips) for peptide sample loading, concentration, and desalting.

Targeted mass spectrometry analysis of the proteins IGF1, IGF2, IBP2, IBP3 and A2GL by blood protein precipitation.

Unlabelled: Biomarker analysis of blood samples by liquid chromatography (LC) mass spectrometry (MS) is extremely challenging due to the high protein concentration range, characterised by abundant proteins that suppress and mask other proteins of lower abundance. This situation is further aggravated when using fast high-throughput methods, which are necessary for analysis of hundreds and thousands of samples in clinical laboratories. The blood proteins IGF1, IGF2, IBP2, IBP3 and A2GL have been proposed as indirect biomarkers for detection of GH administration and as putative biomarkers for breast cancer diagnosis.

KYSS: mass spectrometry data quality assessment for protein analysis and large-scale proteomics.

We introduce the computer tool "Know Your Samples" (KYSS) for assessment and visualisation of large scale proteomics datasets, obtained by mass spectrometry (MS) experiments. KYSS facilitates the evaluation of sample preparation protocols, LC peptide separation, and MS and MS/MS performance by monitoring the number of missed cleavages, precursor ion charge states, number of protein identifications and peptide mass error in experiments. KYSS generates several different protein profiles based on protein abundances, and allows for comparative analysis of multiple experiments.

Depletion of abundant plasma proteins by poly(N-isopropylacrylamide-acrylic acid) hydrogel particles.

Protein and proteome analysis of human blood plasma presents a challenge to current analytical platforms such as mass spectrometry (MS). High abundance plasma proteins interfere with detection of potential protein biomarkers that are often 3-10 orders of magnitude lower in concentration. We report the application of pH-sensitive poly(N-isopropylacrylamide-acrylic acid) hydrogel particles for removal of abundant plasma proteins, prior to proteome analysis by MS.

Growth hormone secretagogues: out of competition.

Growth hormone secretagogues (GHS) constitute a new GH deficiency treatment increasing exponentially in number and improved potency and bioavailability over the last decade. The growth hormone releasing activity makes these compounds attractive for the artificial improvement of the human sports skills, now that recombinant human growth hormone (rhGH) administration is effectively detected. The GHS family is extremely diverse both in number and chemical heterogeneity and keeps growing continuously.

Growth hormone abuse and biological passport: is mannan-binding lectin a complementary candidate?

Objective: In the detection of human growth hormone (GH) abuse, the approach based on altered GH-related biomarkers is also being considered with respect to its application within the context of a biological passport. As a potential biomarker, mannan-binding lectin (MBL), which is reported to respond to recombinant GH (rGH) administration, is evaluated here.

Design: Randomized and single blind and approved by the Ethical Committee (Comité Ético de Investigación Clínica-Instituto Municipal de Asistencia Sanitaria).

Generation of 5 and 17 kDa human growth hormone fragments through limited proteolysis.

Introduction: The reported presence of two fragments of 5 and 17 kDa originating from the 22 kDa human growth hormone (hGH) in blood and tissues, postulated as the sequences AA 1-43 and AA 44-191, has led to the hypothesis of a post-translational proteolytic origin with respect to the abundant 22 kDa variant (AA 1-191). To evaluate this hypothesis, the activity of several endo-proteases on the 22 kDa hGH protein has been evaluated.

Methods: Proteolysis using pepsin, trypsin, V8-protease, proteinase K and thermolysin were explored under several conditions, including incubation time and pH.

Growth hormone in sport: beyond Beijing 2008.

Human growth hormone (hGH) is a protein endogenously produced predominantly by the anterior pituitary gland. Native hGH and, especially, its recombinant analogue (rhGH), used to treat patients with hormone deficiency, are supposed to be abused by athletes searching its anabolic and lipolytic effects. Hence, hGH use has been prohibited for a long time by the sport authorities, but until recently, hGH abuse could not be detected.

Characterisation of the 5 kDa growth hormone isoform.

Objectives: The 5 kDa N-terminal fragment of 43 amino acids of human growth hormone (GH) shows a specific and significant in-vivo insulin-like activity. This isoform can be easily obtained by solid phase synthesis methods. Our objective in this study is to describe this procedure in detail and to provide structural information of the protein.