5 research outputs found
Introducing AAA-MS, a Rapid and Sensitive Method for Amino Acid Analysis Using Isotope Dilution and High-Resolution Mass Spectrometry
Accurate quantification of pure peptides and proteins
is essential for biotechnology, clinical chemistry, proteomics, and
systems biology. The reference method to quantify peptides and proteins
is amino acid analysis (AAA). This consists of an acidic hydrolysis
followed by chromatographic separation and spectrophotometric detection
of amino acids. Although widely used, this method displays some limitations,
in particular the need for large amounts of starting material. Driven
by the need to quantify isotope-dilution standards used for absolute
quantitative proteomics, particularly stable isotope-labeled (SIL)
peptides and PSAQ proteins, we developed a new AAA assay (AAA-MS).
This method requires neither derivatization nor chromatographic separation
of amino acids. It is based on rapid microwave-assisted acidic hydrolysis
followed by high-resolution mass spectrometry analysis of amino acids.
Quantification is performed by comparing MS signals from labeled amino
acids (SIL peptide- and PSAQ-derived) with those of unlabeled amino
acids originating from co-hydrolyzed NIST standard reference materials.
For both SIL peptides and PSAQ standards, AAA-MS quantification results
were consistent with classical AAA measurements. Compared to AAA assay,
AAA-MS was much faster and was 100-fold more sensitive for peptide
and protein quantification. Finally, thanks to the development of
a labeled protein standard, we also extended AAA-MS analysis to the
quantification of unlabeled proteins
MOESM4 of Systematic quantitative analysis of H2A and H2B variants by targeted proteomics
Additional file 4. Details of the SRM transitions for each signature peptide. SRM assay parameters including precursor and fragment ion type, charge state, elution time as well as raw data are provided in Suppl. data. (*) Indicates peptides monitored only in their endogenous form
MOESM5 of Systematic quantitative analysis of H2A and H2B variants by targeted proteomics
Additional file 5. Composition of the mixture of standard peptides
MOESM9 of Systematic quantitative analysis of H2A and H2B variants by targeted proteomics
Additional file 9. Rules used to select or reject peptides using their transition profiles. The validation of the best transitions was performed using a signal-to-noise ratio (>聽5) and a perfect co-elution of the heavy standard peptide with the endogenous peptide. Three fragment ions (F1, F2, and F3) are represented for the heavy and the endogenous peptides. a All fragment ions can be integrated because the heavy and endogenous fragment ions co-elute in the same intensity order. b In that case, only F2 can be integrated because the ratio heavy/endogenous is different for F1 and F3. c The fragment F2 is contaminated by another analyte eluting at a slightly later time; it has to be excluded from the analysis. d Here, the signal-to-noise ratio is below five, no fragment ion can be integrated. e. The endogenous peptide traces do not co-elute with the heavy peptide traces
<i>DIGESTIF</i>: A Universal Quality Standard for the Control of Bottom-Up Proteomics Experiments
In bottom-up mass spectrometry-based
proteomics analyses, variability
at any step of the process, particularly during sample proteolysis,
directly affects the sensitivity, accuracy, and precision of peptide
detection and quantification. Currently, no generic internal standards
are available to control the quality of sample processing steps. This
makes it difficult to assess the comparability of MS proteomic data
obtained under different experimental conditions. Here, we describe
the design, synthesis, and validation of a universal protein standard,
called <i>DIGESTIF</i>, that can be added to any biological
sample. The <i>DIGESTIF</i> standard consists of a soluble
recombinant protein scaffold to which a set of 11 artificial peptides
(iRT peptides) with good ionization properties has been incorporated.
In the protein scaffold, the amino acids flanking iRT peptide cleavage
sites were selected either to favor or hinder protease cleavage. After
sample processing, the retention time and relative intensity pattern
of the released iRT peptides can be used to assess the quality of
sample workup, the extent of digestion, and the performance of the
LC鈥揗S system. Thus, <i>DIGESTIF</i> can be used to
standardize a broad spectrum of applications, ranging from simple
replicate measurements to large-scale biomarker screening in biomedical
applications