Analysis of human serum by liquid chromatography–mass spectrometry: Improved sample preparation and data analysis

Govorukhina, Natalia; Reijmers, Theo H.; Nyangoma, Stephen; Zee, van der Ate; Jansen, Ritsert C.; Bischoff, Rainer

doi:10.1016/j.chroma.2006.02.088

Cited by 66 publications

(64 citation statements)

References 31 publications

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“…Such devices also allow integrating the electrical contact point into the chip, thereby avoiding the need for metal-coated spray needles, which have a limited long-term stability. In the present study, we show that chip-LC-MS compares favorably with a previously used capillary LC-MS (cap-LC-MS) system (1 mm id column) [15] in terms of repeatability for both retention time and peak area. Human serum samples depleted of the six most abundant proteins using a commercially available affinity column followed by trypsin digestion were used to evaluate the chip-LC-MS system.…”

Section: Introductionmentioning

confidence: 64%

“…V5111, Madison, Wisconsin, USA). In the case cytochrome c was spiked into serum, its recovery was shown to be 23.8 6 8.0% after the depletion [15].…”

Section: Sample Preparationmentioning

confidence: 95%

“…All samples were analyzed by LC-MS on the same day. Six peptides from three high-abundance proteins (apolipoprotein A-I (accession number: P02647, Swiss-Prot/Trembl), a-2-macroglobulin precursor (P01023), human complement C3 precursor (P01024)) with m/z values of 756.7, 753.5, 909.6, 682.5, 619.2, and 694.4 m/z [15] were selected to test the analytical repeatability for both cap-LC-MS and chip-LC-MS. First manual integration of peak areas was performed using extracted ion chromatograms (EICs) for m/z values taken at the maximal intensity of each selected peptide peak 6 0.5 m/z, in order to account for shifts occurring in the IT mass analyzer during peak elution. Retention time variation was small in both cases, although the cap-LC-MS system showed a slightly higher SD (mean value of 0.29 min 6 0.15) than chip-LC-MS (mean value of 0.04 min 6 0.01).…”

Section: Repeatabilitymentioning

confidence: 99%

“…Because of these shortcomings nano-LC-MS has found its main application in situations where only a few runs need to be compared such as for protein identification from minute sample amounts. As a result, most biomarker discovery studies use columns of 0.3-1 mm id (flow rates of 1-50 mL/min) for the label-free profiling of complex biological fluids [11,15].…”

Section: Introductionmentioning

confidence: 99%

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Chip‐LC‐MS for label‐free profiling of human serum

et al. 2007

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The discovery of biomarkers in easily accessible body fluids such as serum is one of the most challenging topics in proteomics requiring highly efficient separation and detection methodologies. Here, we present the application of a microfluidics-based LC-MS system (chip-LC-MS) to the label-free profiling of immunodepleted, trypsin-digested serum in comparison to conventional capillary LC-MS (cap-LC-MS). Both systems proved to have a repeatability of approximately 20% RSD for peak area, all sample preparation steps included, while repeatability of the LC-MS part by itself was less than 10% RSD for the chip-LC-MS system. Importantly, the chip-LC-MS system had a two times higher resolution in the LC dimension and resulted in a lower average charge state of the tryptic peptide ions generated in the ESI interface when compared to cap-LC-MS while requiring approximately 30 times less (~5 pmol) sample. In order to characterize both systems for their capability to find discriminating peptides in trypsin-digested serum samples, five out of ten individually prepared, identical sera were spiked with horse heart cytochrome c. A comprehensive data processing methodology was applied including 2-D smoothing, resolution reduction, peak picking, time alignment, and matching of the individual peak lists to create an aligned peak matrix amenable for statistical analysis. Statistical analysis by supervised classification and variable selection showed that both LC-MS systems could discriminate the two sample groups. However, the chip-LC-MS system allowed to assign 55% of the overall signal to selected peaks against 32% for the cap-LC-MS system.

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Section: Introductionmentioning

confidence: 64%

“…V5111, Madison, Wisconsin, USA). In the case cytochrome c was spiked into serum, its recovery was shown to be 23.8 6 8.0% after the depletion [15].…”

Section: Sample Preparationmentioning

confidence: 95%

Section: Repeatabilitymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Chip‐LC‐MS for label‐free profiling of human serum

et al. 2007

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“…Moreover, the high cost, the scarce selectivity, or long operation times, impose the development of new depletion systems based on multiple affinity columns [30,31]. The latter are characterized by the presence of specific antibodies derivatized columns which specifically and selectively deplete a number of high-abundant proteins in serum plus their proteolytic products and molecular forms [32][33][34][35][36][37]. The selection of a methodological approach providing optimal reduction of the serum dynamic range with high reproducibility represents a critical point for the translation of MALDI serum profiling to clinical laboratories.…”

Section: Introductionmentioning

confidence: 99%

Setup for human sera MALDI profiling: The case of rhEPO treatment

et al. 2011

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The implementation of high-throughput technologies based on qualitative and quantitative methodologies for the characterization of complex protein mixtures is increasingly required in clinical laboratories. MALDI profiling is a robust and sensitive technology although the serum high dynamic range imposes a major limitation hampering the identification of less abundant species decreasing the quality of MALDI profiling. A setup to improve these parameters has been performed for recombinant human erythropoietin (rhEPO) monitoring in serum, analyzing the effects of two commercially available columns (MARS Hu7 and Hu14) for immunodepletion, and two matrices (acyano-4-hydroxycinnamic acid and 2 0 ,4 0 -dihydroxyacetophenone) for peak quality improvement. The immunodepletion capability of both columns was determined by 2-D DIGE, which precisely revealed the efficacy of Hu14 in protein removal and the serum dynamic range decrement. In addition, the type of matrix, the sample dilution, and the efficacy of optimized parameters were used for serum profiling of ten healthy subjects before and after rhEPO treatment. The principal component analysis indicates that a combination of Hu14 column and 2 0 ,4 0 -dihydroxyacetophenone matrix increases data quality allowing the discrimination between treated and untreated samples, making serum MALDI profiling suitable for clinical monitoring of rhEPO.

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Tetrachlorinated Tetraazaperopyrenes (TAPPs): Highly Fluorescent Dyes and Semiconductors for Air‐Stable Organic n‐Channel Transistors and Complementary Circuits

Martens

Zschieschang

Wadepohl

et al. 2012

Chemistry A European J

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A range of 2,9-perfluoroalkyl-substituted tetraazaperopyrene (TAPP) derivatives (1-5) was synthesised by reacting 4,9-diamino-3,10-perylenequinone diimine (DPDI) with the corresponding carboxylic acid chloride or anhydride in the presence of a base. The reaction of compounds 1-4 with dichloroisocyanuric acid (DIC) in concentrated sulphuric acid resulted in the fourfold substitution of the tetraazaperopyrene core, yielding the 2,9-bisperfluoroalkyl-4,7,11,14-tetrachloro-1,3,8,10-tetraazaperopyrenes 6-9, respectively. The optical and electrochemical data demonstrate the drastic influence of the core substitution on the properties. All compounds are highly luminescent (fluorescence quantum yields of up to Φ=0.8). The LUMO energies of the tetrachlorinated TAPP derivatives (determined by cyclic voltammetry and computed by DFT calulations) were found to be below -4 eV. In the course of this work the performance of TAPP derivatives in organic thin-film transistors (TFTs) was investigated, and their n-channel characteristics with field-effect mobilities of up to 0.14 cm(2) V(-1) s(-1) and an on/off current ratio of >10(6) were confirmed. Long-term stabilities of 3-4 months under ambient conditions of the devices were established. Complementary inverters and ring oscillators with n-channel TFTs based on compound 8 and p-channel TFTs based on dinaphtho-[2,3-b:2',3'-f]thieno[3,2-b]thiophene (DNTT) were fabricated on a glass substrate.

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Analysis of human serum by liquid chromatography–mass spectrometry: Improved sample preparation and data analysis

Cited by 66 publications

References 31 publications

Chip‐LC‐MS for label‐free profiling of human serum

Chip‐LC‐MS for label‐free profiling of human serum

Setup for human sera MALDI profiling: The case of rhEPO treatment

Tetrachlorinated Tetraazaperopyrenes (TAPPs): Highly Fluorescent Dyes and Semiconductors for Air‐Stable Organic n‐Channel Transistors and Complementary Circuits

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