not labeled simultaneously, reagents dissolved in anhydrous
acetonitrile can be stored at À20
C for <1 week and should
be warmed to room temperature before opening, according to
Thermo Fisher Scientific protocol.
15. No further additional desalting step is necessary if peptide
fractionation is carried out using pH Reversed-Phase Peptide
Fractionation as proposed here. A desalting step might be
necessary if other pre-fractionation methods are used or for
applications where fractionation is not required.
16. For peptide fractionation step, the spin columns available in the
kit accept between 10 and 100 μg of peptides in a maximum
volume of 300 μL. In our experiments, we have worked with a
total of 30 μg (0.1 μg/μL) of TMT-labeled peptides. At the
end of the fractionation procedure, and for each peptide fraction, we split the final eluted volume in two microtubes (replicates) with the same volume each (~150 μL). At the end, for
each of the eight fractions, there will be about 1.5–2.0 μg of
peptides per replicate (two per fraction), a peptide amount that
in our experience allows obtaining good resolution and results
from LC–MS/MS analysis using a Thermo Fisher Scientific
LTQ-Orbitrap Elite device under conditions described in this
chapter.
17. For peptide quantification using isobaric tags, such as TMT
and iTRAQ, that allows multiplexing several samples in the
same experiment, high-resolution mass spectrometers are necessary. A major drawback of conventional quantitative shotgun
proteomics based on MS2 analysis in data-dependent acquisition approach (DDA) is the non-reliable quantification of some
peptides due to well-known problem of co-isolation of similar
peptides (similar m/z values) from MS1 spectrum, thus contaminating reporter ion signals obtained from MS2 analysis of
target peptides, leading to what has been called as ratio distortion (ratios of quantification of peptides between different
samples tend to be compressed to 1:1). Despite peptide fractionation should contribute to alleviate this problem, a more
effective way to work out this limitation is to proceed with an
additional fragmentation and isolation step producing MS3
spectrum, more concretely using a method known as MultiNotch MS3. However, this additional MS3 step is not exempt
from paying a cost, that is, lowering the number of quantifiable
peptides per experiment. Last-generation mass spectrometers
contain important technological advances that have aided to
polish this further limitation and improving their resolution,
precision, and accuracy. An alternative strategy is working in
data-independent acquisition approach (DIA). For a thoughtful and update review of this issue, authors recommend the
98
Angel P. Diz and Paula Sa ´ nchez-Marı ´n
acetonitrile can be stored at À20
C for <1 week and should
be warmed to room temperature before opening, according to
Thermo Fisher Scientific protocol.
15. No further additional desalting step is necessary if peptide
fractionation is carried out using pH Reversed-Phase Peptide
Fractionation as proposed here. A desalting step might be
necessary if other pre-fractionation methods are used or for
applications where fractionation is not required.
16. For peptide fractionation step, the spin columns available in the
kit accept between 10 and 100 μg of peptides in a maximum
volume of 300 μL. In our experiments, we have worked with a
total of 30 μg (0.1 μg/μL) of TMT-labeled peptides. At the
end of the fractionation procedure, and for each peptide fraction, we split the final eluted volume in two microtubes (replicates) with the same volume each (~150 μL). At the end, for
each of the eight fractions, there will be about 1.5–2.0 μg of
peptides per replicate (two per fraction), a peptide amount that
in our experience allows obtaining good resolution and results
from LC–MS/MS analysis using a Thermo Fisher Scientific
LTQ-Orbitrap Elite device under conditions described in this
chapter.
17. For peptide quantification using isobaric tags, such as TMT
and iTRAQ, that allows multiplexing several samples in the
same experiment, high-resolution mass spectrometers are necessary. A major drawback of conventional quantitative shotgun
proteomics based on MS2 analysis in data-dependent acquisition approach (DDA) is the non-reliable quantification of some
peptides due to well-known problem of co-isolation of similar
peptides (similar m/z values) from MS1 spectrum, thus contaminating reporter ion signals obtained from MS2 analysis of
target peptides, leading to what has been called as ratio distortion (ratios of quantification of peptides between different
samples tend to be compressed to 1:1). Despite peptide fractionation should contribute to alleviate this problem, a more
effective way to work out this limitation is to proceed with an
additional fragmentation and isolation step producing MS3
spectrum, more concretely using a method known as MultiNotch MS3. However, this additional MS3 step is not exempt
from paying a cost, that is, lowering the number of quantifiable
peptides per experiment. Last-generation mass spectrometers
contain important technological advances that have aided to
polish this further limitation and improving their resolution,
precision, and accuracy. An alternative strategy is working in
data-independent acquisition approach (DIA). For a thoughtful and update review of this issue, authors recommend the
98
Angel P. Diz and Paula Sa ´ nchez-Marı ´n