4. You can also see https://www.spectroscopyeurope.com/arti
cle/practical-guide-sample-preparation-liquid-chromatogra
phy-tandem-mass-spectrometry-clinical for more information
about other sample preparation protocols.
5. To perform the correct protein quantification, it is necessary to
select an adequate measuring method. In this sense, for protein
samples with urea or thiourea, compatible methods are necessary to perform the quantification. One kit that is suitable for
the major protein extraction buffer is RDDC (Biorad).
6. In order to improve the efficiency of the precipitation, the time
of precipitation can be increased, and it is possible to perform
overnight precipitation.
7. The LC and autosampler methods will vary between labs, and
the gradient lengths will vary depending on the complexity of
the samples. Typically, for complex mixtures, a gradient of
5–35% of solution B over 90–120 min is suitable, and for less
complex samples (i.e., immunoprecipitations, purified proteins), shorter gradients between 30 and 60 min may be
sufficient.
8. A number of survey scans used in a typical DDA/IDA method
on a TripleTOF
® 6600+ system uses 20 candidate ions,
although this value can be modified.
9. TripleTOF
® 6600+ can go up to 2250 m/z. The larger mass
range is especially important when doing large protein modifications such as glycoproteomics and sumoylations or when
using alternative proteolytic methods that produce larger peptides (i.e., Lys-C, CNBr).
10. Sciex software can be downloaded at http://www.absciex.
com/downloads/software-downloads.
11. FDR threshold is usually selected in 1%.
References
1. Anjo SI, Santa C, Manadas B (2017)
SWATH-MS as a tool for biomarker discovery: from basic research to clinical applications. Proteomics 17:3–4
2. Aebersold R, Mann M (2016) Massspectrometric exploration of proteome structure and function. Nature 537:347–355
3. Darling AL, Uversky VN (2018) Intrinsic disorder and posttranslational modifications: the
darker side of the biological dark matter.
Front Genet 9:1–18
4. FDA-NIH Biomarker Working Group (2016)
BEST (Biomarkers, EndpointS, and other
Tools) Resource. Food and Drug Administration, Silver Spring, MD
5. Fu ¨ze ´ry AK, Levin J, Chan MM, Chan DW
(2013) Translation of proteomic biomarkers
into FDA approved cancer diagnostics: issues
and challenges. Clin Proteomics 10:1
6. Pavlou MP, Diamandis EP, Blasutig IM
(2013) The long journey of cancer biomarkers from the bench to the clinic. Clin Chem
59:147–157
7. Anderson NL (2010) The clinical plasma proteome: a survey of clinical assays for proteins
in plasma and serum. Clin Chem 56:177–185
8. Vidova V, Spacil Z (2017) A review on mass
spectrometry-based quantitative proteomics:
targeted and data independent acquisition.
Anal Chim Acta 964:7–23
SWATH-MS Protocols in Human Diseases
135
cle/practical-guide-sample-preparation-liquid-chromatogra
phy-tandem-mass-spectrometry-clinical for more information
about other sample preparation protocols.
5. To perform the correct protein quantification, it is necessary to
select an adequate measuring method. In this sense, for protein
samples with urea or thiourea, compatible methods are necessary to perform the quantification. One kit that is suitable for
the major protein extraction buffer is RDDC (Biorad).
6. In order to improve the efficiency of the precipitation, the time
of precipitation can be increased, and it is possible to perform
overnight precipitation.
7. The LC and autosampler methods will vary between labs, and
the gradient lengths will vary depending on the complexity of
the samples. Typically, for complex mixtures, a gradient of
5–35% of solution B over 90–120 min is suitable, and for less
complex samples (i.e., immunoprecipitations, purified proteins), shorter gradients between 30 and 60 min may be
sufficient.
8. A number of survey scans used in a typical DDA/IDA method
on a TripleTOF
® 6600+ system uses 20 candidate ions,
although this value can be modified.
9. TripleTOF
® 6600+ can go up to 2250 m/z. The larger mass
range is especially important when doing large protein modifications such as glycoproteomics and sumoylations or when
using alternative proteolytic methods that produce larger peptides (i.e., Lys-C, CNBr).
10. Sciex software can be downloaded at http://www.absciex.
com/downloads/software-downloads.
11. FDR threshold is usually selected in 1%.
References
1. Anjo SI, Santa C, Manadas B (2017)
SWATH-MS as a tool for biomarker discovery: from basic research to clinical applications. Proteomics 17:3–4
2. Aebersold R, Mann M (2016) Massspectrometric exploration of proteome structure and function. Nature 537:347–355
3. Darling AL, Uversky VN (2018) Intrinsic disorder and posttranslational modifications: the
darker side of the biological dark matter.
Front Genet 9:1–18
4. FDA-NIH Biomarker Working Group (2016)
BEST (Biomarkers, EndpointS, and other
Tools) Resource. Food and Drug Administration, Silver Spring, MD
5. Fu ¨ze ´ry AK, Levin J, Chan MM, Chan DW
(2013) Translation of proteomic biomarkers
into FDA approved cancer diagnostics: issues
and challenges. Clin Proteomics 10:1
6. Pavlou MP, Diamandis EP, Blasutig IM
(2013) The long journey of cancer biomarkers from the bench to the clinic. Clin Chem
59:147–157
7. Anderson NL (2010) The clinical plasma proteome: a survey of clinical assays for proteins
in plasma and serum. Clin Chem 56:177–185
8. Vidova V, Spacil Z (2017) A review on mass
spectrometry-based quantitative proteomics:
targeted and data independent acquisition.
Anal Chim Acta 964:7–23
SWATH-MS Protocols in Human Diseases
135