The use of turbulent flow chromatography for rapid, on-line analysis of tryptic digests

Article abstract of DOI:10.1002/rcm.7371

Rationale Following digestion of proteins with trypsin, digests are typically subjected to further 'clean-up' prior to liquid chromatography/mass spectometry (LC/MS) analysis, in order to reduce the complexity of the digested matrix, as well as helping to remove residual denaturants and reduction/alkylation reagents prior to injection onto the analytical HPLC column. Often, this is carried out using off-line techniques, and is not ideally suited to high-throughput workloads, for example in clinical laboratories. Methods Bovine serum albumin (BSA) was used as a model protein. Following denaturation with urea, reduction/alkylation, and digestion with trypsin, the analytical recovery of a selection of proteotypic BSA peptides was assessed using a two-dimensional, turbulent flow chromatography method. Peptides were identified using a Q Exactive mass spectometer operating in full-scan mode. Results Total analysis time (including the on-line sample clean-up) was 15 min per injection. Aside from the most hydrophilic peptide selected, ATEEQLK, recovery using the turbulent flow chromatography systems was greater than 30% for all remaining peptides (N=17), and exceeded 50% for 12 of the 18 peptides studied. There was a broad correlation between the hydrophobicity factor and the observed recovery. Conclusions This study suggests that turbulent flow chromatography offers a rapid, on-line alternative to solid-phase extraction for the analysis of peptide digests by LC/MS. A wide range of column chemistries are available, and the technique can be further optimised for analyses which are targetted to specific peptides. As with turbulent flow chromatography for small-molecule workflows, this approach may be ideally suited to high-throughput applications, such as those which are emerging from within clinical laboratories.

Full text of DOI:10.1002/rcm.7371

Research Article
Received: 19 June 2015
Revised: 19 August 2015
Accepted: 21 August 2015
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2015, 29, 2140–2146
(wileyonlinelibrary.com) DOI: 10.1002/rcm.7371
The use of turbulent flow chromatography for rapid, on-line
analysis of tryptic digests
2
3
L. Couchman1,2 , D. J. L. Jones and C. F. Moniz
*
1Viapath Analytics, Department of Clinical Biochemistry, King’s College Hospital, Denmark Hill, London SE5 9RS, UK
2Department of Cancer Studies, Leicester Royal Infirmary, University of Leicester, Leicester LE2 7LX, UK
3Department of Clinical Biochemistry, King’s College Hospital, Denmark Hill, London SE5 9RS, UK
RATIONALE: Following digestion of proteins with trypsin, digests are typically subjected to further ’clean-up’ prior to
liquid chromatography/mass spectometry (LC/MS) analysis, in order to reduce the complexity of the digested matrix, as
well as helping to remove residual denaturants and reduction/alkylation reagents prior to injection onto the analytical
HPLC column. Often, this is carried out using off-line techniques, and is not ideally suited to high-throughput work-
loads, for example in clinical laboratories.
METHODS: Bovine serum albumin (BSA) was used as a model protein. Following denaturation with urea,
reduction/alkylation, and digestion with trypsin, the analytical recovery of a selection of proteotypic BSA peptides
was assessed using a two-dimensional, turbulent flow chromatography method. Peptides were identified using a Q
Exactive™ mass spectometer operating in full-scan mode.
RESULTS: Total analysis time (including the on-line sample clean-up) was 15 min per injection. Aside from the most
hydrophilic peptide selected, ATEEQLK, recovery using the turbulent flow chromatography systems was greater than
30% for all remaining peptides (N = 17), and exceeded 50% for 12 of the 18 peptides studied. There was a broad correlation
between the hydrophobicity factor and the observed recovery.
CONCLUSIONS: This study suggests that turbulent flow chromatography offers a rapid, on-line alternative to solid-phase
extraction for the analysis of peptide digests by LC/MS. A wide range of column chemistries are available, and the technique
can be further optimised for analyses which are targetted to specific peptides. As with turbulent flow chromatography for
small-molecule workflows, this approach may be ideally suited to high-throughput applications, such as those which are
emerging from within clinical laboratories. Copyright © 2015 John Wiley & Sons, Ltd.
In recent years, interest in the measurement of clinically relevant
proteins and peptides using liquid chromatography/mass
spectrometry (LC/MS) has gained momentum. Proteomics is
still largely restricted to the research environment, but the
specificity and sensitivity provided by MS, and hence the
ability to identify protein isoforms and variants, is an attractive
prospect for translation to the clinical laboratory. The
International Federation of Clinical Chemistry (IFCC) has
convened a working group dedicated to clinical quantitative
mass spectrometry proteomics.[1] Furthermore, those more
accustomed to LC/MS for targetted small-molecule workflows
can now develop quantitative protein assays owing to the
number of protein quantitation strategies possible, the availability
of stable-isotope-labelled proteins and peptides,[2,3] and the
accessibility of vast on-line protein/peptide databases/spectral
libraries.[4]
(typically trypsin) is required prior to MS analysis of surrogate
proteotypic peptides.[1–3] The digestion step in such workflows
can be carried out either directly on the biological matrix itself,
or following some form of sample ‘clean-up’ (e.g. removal of
high-abundance proteins or immunoenrichment) prior to
digestion of remaining proteins/peptides.
Post-digestion sample ’clean-up’ typically uses solid-phase
extraction (SPE) techniques. This reduces the complexity of
the digested matrix, as well as helping to remove residual
denaturants and reduction/alkylation reagents prior to
injection onto the analytical HPLC column. These procedures
are often carried out ’off-line’ using either 96-well plate-based
SPE systems, or with modified pipette tips containing silica
beds,[6,7] and is primarily based on reversed-phase interactions
with C18-modified phases. Some groups have also used
mixed-mode cation-exchange systems or two-dimensional
SPE approaches for specific target peptides.[8]
Turbulent flow chromatography is a rapid, on-line sample
clean-up technique that has been applied in the field of
quantitative small-molecule analysis for many years.[9] This
approach uses high eluent flow rates, and narrow columns
packed with large (>50 μm), irregularly shaped particles to
induce turbulent flow. This allows separation of large
biomolecules from smaller analytes of interest by way of a
’pseudo-size-exclusion’ mechanism, which results from the
Whilst analysis of ’intact’ proteins/peptides is possible, and
arguably preferable for some target proteins (e.g. insulin),[5]
for many others a digestion step using a suitable protease
* Correspondence to: L. Couchman, Toxicology Unit, Department
of Clinical Biochemistry (Viapath Analytics), King’s College
Hospital, Denmark Hill, London SE5 9RS, UK.
E-mail: lewis.couchman@nhs.net
Rapid Commun. Mass Spectrom. 2015, 29, 2140–2146
Copyright © 2015 John Wiley & Sons, Ltd.

Turbulent flow chromatography for analysis of tryptic digests
investigated the performance of turbulent flow chromatography
(TurboFlow™ technology, ThermoFisher Scientific) for the
automated, on-line analysis of tryptic digests using an aqueous
solution of bovine serum albumin (BSA) as a model protein.
EXPERIMENTAL
Chemicals and reagents
Ammonium bicarbonate, urea, DL-dithiothrietol (DTT, as solid),
iodoactemide (IAA), calcium chloride, tris(hydroxymethyl)
aminomethane (Trizma®, 99.9%), bovine serum albumin (BSA),
TCPK-treated trypsin (from bovine pancreas) and LC/MS grade
acetonitrile were all purchased from Sigma-Aldrich (Poole, UK).
MS grade formic acid and trifluoroacetic acid (TFA) were from
Fluka (Poole, UK). Ammonia solution (>25%), isopropanol,
acetone and glacial acetic acid were from VWR (all AnalaR
grade, Lutterworth, UK). Water was de-ionised (>12 mΩ, Purite,
Thame, UK). A 1 mol/L aqueous stock solution of DTT was
prepared and stored in portions at À20 °C. Reducing solution
(8 mol/L urea, 300 mmol/L tris, and 10 mmol/L DTT in 2.5 %
(v/v) isopropanol in deionised water) was prepared
immediately prior to digestion. IAA solution (200 mmol/L in
100 mmol/L aqueous ammonium bicarbonate) was prepared
Figure 1. TurboFlow valve arrangement showing (a) the
valve positions during loading and re-equilibration steps
and (b) the transfer/focusing step.
fact that larger molecules diffuse more slowly in solution than
small molecules, and so do not have sufficient time within the
turbulent flow column to enter the particle pores and interact
with the stationary phase chemistry. In this study, we have
Table 1. TurboFlow method summary
Loading pump (MCX2/C18 XL)
Eluting pump (XDB-C18)
Step
Time (s)
Flow rate (mL/min)
%A
%B
%C
%D
Tee
Loop
Flow rate (mL/min)
%A
%B
1
2
3
4
5
6
7
8
9
30
40
500
1
60
1
60
80
40
1.50
0.10
0.50
0.50
0.50
0.50
1.00
1.00
1.50
100
-
-
-
-
Out
In
Out
In
0.30
0.90
0.30
0.30
0.40
0.40
0.40
0.40
0.30
98
98
40
-
2
2
100
-
-
-
-
-
-
-
100
-
100
100
100
-
-
-
-
Out
Out
Out
Out
Out
Out
Out
In
60
100
100
2
-
-
Out
Out
Out
In
Out
Out
-
-
-
-
-
98
98
98
98
40
-
60
-
2
100
100
2
-
-
2
Table 2. BSA tryptic peptides, m/z values and hydrophobicity factors
m/z (3 most intense isotopes from
Krokhin
hydrophobicity factor
Peptide sequence
the most abundant charge state)
Sequence location
1
ATEEQLK
409.7163, 410.2178, 410.7191
352.2085, 352.7099, 353.2111
443.7113, 444.2127, 444.7140
487.7325, 488.2340, 488.7353
395.2395, 395.7409, 396.2422
461.7477, 462.2491, 462.7505
653.3617, 653.8631, 654.3645
501.7951, 502.2966, 502.7979
464.2504, 464.7518, 465.2532
642.3589, 642.8604, 643.3618
756.4250, 756.9265, 757.4278
582.3190, 582.8204, 583.3218
507.8133, 508.3148, 508.8161
740.4014, 740.9028, 741.4042
630.3138, 630.6481, 630.9824
978.4835, 978.9849, 979.4863
700.3499, 700.8514, 701.3522
784.3750, 784.8765, 785.3779
6.14
7.47
538-544
188-194
107-114
13-20
233-239
225-232
378-388
574-583
137-143
337-347
414-427
42-51
525-533
397-409
145-159
295-312
545-556
323-335
2
VLASSAR
3
DDSPDLPK
15.56
15.92
17.82
22.22
22.27
25.47
28.22
29.97
30.00
31.99
35.35
38.67
40.20
43.27
45.10
50.58
4
DLGEEHFK
5
LVTDLTK
6
AEFVEVTK
7
HLVDEPQNLIK
LVVSTQTALA
YLYEIAR
8
9
10
11
12
13
14
15
16
17
18
HPEYAVSVLLR
VPQVSTPTLVEVSR
LVNETEFAK
QTALVELLK
LGEYGFQNALIVR
HPYFYAPELLYYANK
DAIPENLPPLTADFAEDK
TVMENFVAFVDK
DAFLGSFLYEYSR
Rapid Commun. Mass Spectrom. 2015, 29, 2140–2146
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L. Couchman, D. J. L. Jones and C. F. Moniz
Figure 2. (A) Example total ion chromatograms for (i) LX and (ii) TX injections of BSA digests. (B) Extracted ion chromatograms
( 5 ppm) and averaged mass spectra for LX and TX injections of BSA digest showing the peptide LVNETEFAK. Shown
separately are the three most abundant isotopes for the most abundant charge state (2+; 582.3189, 582.8204, 583.3218).
Chromatograms are scaled relative to the largest peak (582.3189 m/z).
wileyonlinelibrary.com/journal/rcm
Copyright © 2015 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2015, 29, 2140–2146

Turbulent flow chromatography for analysis of tryptic digests
and stored in the dark in amber glass vials at 2–8 °C. Dilution
buffer (50 mmol/L tris and 5 mmol/L calcium chloride,
aqeuous) was prepared and stored at 2–8 °C. The required
volume of trypsin solution (0.5 μg/μL in aqueous 25 mmol/L
acetic acid) was prepared immediately prior to digestion. For this
study, an aqueous solution was prepared containing 50 g/L BSA.
C, and the elution solvent loop re-filled for the subsequent
injection. The whole system was then re-equilibrated prior to
the next injection. The gradient elution and valve-switching
profile are summarised in Table 1. Total analysis time was
15 min. Eluent flow was diverted to waste for the first and
final 2 min of each analysis, with tandem mass spectrometric
(MS/MS) data only acquired for 12 min.
Detection was carried out in positive ionisation mode using
heated electrospray (H-ESI) ionisation (spray voltage 4.5 kV;
temperatures: vaporiser 200 °C; capillary 320 °C; auxiliary,
sheath, and sweep gases 50, 5 and 0 (arbitrary units)
respectively, S-lens voltage 100 V). Data were acquired in
full-scan mode (70,000 resolution, range m/z 300–1500,
maximum injection time 200 ms, AGC target 3 × 10e6). For
this study, no fragmentation (MS2) data were acquired.
Instrumentation and conditions
An Aria Transcend™ TLX-II system (ThermoFisher Scientific,
San Jose, CA, USA) consisting of four Accela™ 1250 high-
pressure quaternary pumps, valve interface module (VIM),
and CTC PAL autosampler was used with a Q Exactive™
high-resolution mass spectrometer (ThermoFisher Scientific,
Bremen, Germany). The autosampler tray was maintained
at 10 °C. Instrument control was performed using XCalibur™
software (version 2.2, ThermoFisher Scientific). System eluents
were as follows: (Loading Pump Eluent A) 0.1% (v/v) formic
acid and 0.05% (v/v) TFA in deionised water, (Eluting Pump
Eluent A) 0.1% (v/v) formic acid in deionised water,
(Loading/Eluting Pump Eluents B) 0.1% (v/v) formic acid in
acetonitrile, (Loading Pump Eluent C) 1+2+2 (v/v/v) acetone,
acetonitrile and isopropanol (’Magic Mix’), and (Loading Pump
Eluent D) 2% (v/v) ammonia solution in deionised water.
Prepared sample digests (50 μL) were injected onto the
TurboFlow™ columns (Cyclone MCX-2 and C18-XL
connected in series, each 50 × 0.5 mm i.d.) under turbulent
flow (1.5 mL/min). Retained peptides were subsequently
eluted from the TurboFlow column using elution solvent
(100 μL, 2+3 (v/v) Loading Pump Eluent B:Loading Pump
Eluent D, stored in a holding loop), and focused through a
tee-piece onto the analytical HPLC column (Fig. 1). Peptides
were separated using a Zorbax™ Eclipse XDB-C18 HPLC
column (Agilent Technologies, 1.8 μm average particle size,
50 × 4.6 mm i.d.) maintained at 50 °C. During gradient elution
(total flow rate 300 μL/min) of the analytes from the analytical
HPLC column, the TurboFlow column was washed with
Loading Pump Eluent B, followed by Loading Pump Eluent
Digestion procedure
Digestion was carried out in 2 mL Lo-Bind tubes (Eppendorf,
Stevenage, UK). Freshly prepared reducing solution (60 μL)
was added to BSA solution (50 g/L, 20 μL) and the tube then
capped and incubated at 37 °C for 1 h. The tube was
centrifuged briefly (10,000 rpm, 30 s) to remove condensation
from the tube cap, and allowed to cool to room temperature.
IAA solution (10 μL) was then added, the tube re-capped, and
the mixture incubated at room temperature in the dark for
30 min. The mixture was diluted with dilution buffer (350 μL)
and vortex mixed (5 s) to reduce the urea concentration prior
to digestion. Freshly prepared trypsin solution was then added
(trypsin:BSA ratio 1:12.5), and the tubes were re-capped and
vortex mixed (5 s), and incubated (37 °C, 4 h). Digestion was
quenched by the addition of formic acid (6 μL), and digests
were diluted (1+3, v/v) with Loading Pump Eluent A in
2 mL glass autosampler vials prior to analysis.
Peptide targetting and data processing/analysis
Eighteen BSA tryptic peptides were identified following in
silico digestion (trypsin, assuming no missed cleavages) of
9.00
y = 0.0677x + 5.5288
R² = 0.9686
8.50
8.00
7.50
7.00
6.50
6.00
5.50
DAFLGSFLYEYSR
TVMENFVAFVDK
DAIPENLPPLTADFAEDK
HPYFYAPELLYYANK
LGEYGFQNALIVR
QTALVELLK
LVNETEFAK
VPQVSTPTLVEVSR
HPEYAVSVLLR
YLYEIAR
LVVSTQTALA
HLVDEPQNLIK
AEFVEVTK
LVTDLTK
DLGEEHFK
DDSPDLPK
ATEEQLK
5
15
25
35
45
55
Krokhin Hydrophobicity Factor
Figure 3. Observed chromatographic retention time (min) compared to the
calculated hydrophobicity factor (from Pinpoint software) for the 17 BSA peptides.
Rapid Commun. Mass Spectrom. 2015, 29, 2140–2146
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L. Couchman, D. J. L. Jones and C. F. Moniz
the amino acid sequence imported sequence from Uniprot[10]
(entry [position] P02769 [25–607]) using PinPoint™ software
(version 1.3, ThemoFisher Scientific). For this study, peptides
containing cysteine residues, and those less than 7 amino
acids in length, were excluded (Table 2).
For all data processing and calculation of recovery,
full-scan data were filtered ( 5 ppm exclusion window)
for the m/z values of interest for each tryptic peptide
(cumulative peaks are for the top 3 most intense isotopes
for the most abundant charge state). No chromatographic
smoothing was performed prior to peak integration.
Recovery (%) for each peptide was calculated by comparison
of the mean integrated peak areas (N = 10 injections of each)
of direct laminar flow (LX) and turbulent flow (TX) injections
of BSA digests.
RESULTS AND DISCUSSION
Example total ion current (TIC) chromatograms and
extracted ion chromatograms for representative peptides
(LX and TX injections) are shown in Fig. 2. There was
excellent correlation between the observed peptide retention
times with the Krokhin hydrophobicity factors (calculated
within Pinpoint software, Fig. 3).[11] Repeat injection of
prepared BSA digests (N = 10) showed excellent peak area
reproducibility for both LX (% RSD for all peptides 1.4–6.9%)
and TX (1.0–5.9%) injections (Table 3). The peptide VLASSAR
was not observed in any chromatogram (LX or TX injections)
following the 4 h digestion protocol described.
Aside from the most hydrophilic peptide, ATEEQLK
(hydrophobicity factor 6.14), TurboFlow recovery was greater
Table 3. Summary recovery data for selected BSA tryptic peptides
Retention
time (min)
Krokhin
hydrophobicity factor
Precision
(%RSD, N = 10), LX
Precision
(%RSD, N = 10), TX
Recovery
(%)
Peptide sequence
1
ATEEQLK
5.80
-
6.14
(7.47)
15.56
15.92
17.82
22.22
22.27
25.47
28.22
29.97
30.00
31.99
35.35
38.67
40.20
43.27
45.10
50.58
6.24
-
2.30
-
15.3
-
2
VLASSAR
3
DDSPDLPK
6.54
6.51
6.86
6.93
7.07
7.66
7.31
7.64
7.52
7.72
8.12
8.17
8.08
8.31
8.57
8.94
3.85
1.63
3.11
3.15
2.22
2.76
1.70
2.20
2.25
1.76
1.43
1.56
6.85
3.10
1.79
5.85
4.50
4.99
2.77
3.51
1.44
5.56
2.36
2.36
1.03
1.86
1.88
2.81
5.51
5.85
2.91
4.88
30.5
27.7
39.5
36.5
69.8
84.0
53.9
69.1
76.0
70.5
69.9
78.3
99.2
54.5
70.5
84.6
4
DLGEEHFK
5
LVTDLTK
6
AEFVEVTK
7
HLVDEPQNLIK
LVVSTQTALA
YLYEIAR
8
9
10
11
12
13
14
15
16
17
18
HPEYAVSVLLR
VPQVSTPTLVEVSR
LVNETEFAK
QTALVELLK
LGEYGFQNALIVR
HPYFYAPELLYYANK
DAIPENLPPLTADFAEDK
TVMENFVAFVDK
DAFLGSFLYEYSR
Figure 4. Correlation between the calculated hydrophobicity factor (from
Pinpoint software) for the 17 BSA peptides and the TurboFlow recovery
(% mean peak area, TX vs LX injections).
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Copyright © 2015 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2015, 29, 2140–2146

Turbulent flow chromatography for analysis of tryptic digests
than 30% for all peptides, and was greater than 50% for 12 of
the 18 peptides studied (Table 3). There was a broad
correlation between the hydrophobicity factor and the observed
recovery (Fig. 4).
use of stable-isotope-labelled internal standards is always
advocated where possible. Owing to the inevitable differences
in peptide recovery (as observed in this study, but also equally
likely with SPE-based methods), the use of isotope-labelled
standards for each of the targeted tryptic peptides should be
recommended, and recovery studies carried out during
method validation to ensure similarity between the labelled
and unlabelled peptides. Likewise, careful optimisation of
the digestion method is still a requirement – ongoing
developments of approaches to speed-up trypsin digestion
protocols (e.g. using thermal-cycling)[14] will be an important
future consideration for high-throughput workflows.
However, as with SPE workflows, TurboFlow methods can
be optimised to ensure maximal recovery of the target peptide
for analyses which require a specific tryptic peptide to be
quantified (e.g. in the quantitation of protein isoforms and
variants). Examples could include manipulation of the
Loading Eluent pH, variation in the TFA concentration, and
optimisation of the elution loop contents. For instance, the
recovery of more hydrophobic peptides may be increased by
increasing the organic solvent content in the elution loop. It
should be noted that this may affect the chromatographic
peak shape of more hydrophilic peptides as there may be less
analyte focusing during the transfer step, and so often a
compromise must be reached in cases where multiple peptides
with different retention characteristics are to be simultaneously
analysed.
CONCLUSIONS
Although a preliminary study based on a single model
protein, our data suggest that turbulent flow chromatography
may be useful for the on-line analysis of tryptic digests, and
thus could be well suited to high-throughput, targetted,
peptide-based protein analyses. Compared to off-line SPE
techniques, this approach is rapid and fully automated with
regards to the valve-switching setup. Even when compared
with methods where tryptic digests are analysed directly by
LC/MS with no additional sample pre-treatment, since the
TurboFlow separation process occurs very rapidly (within
20–30 s) following injection, there is very little impact on the
total analysis time. Furthermore, the physics of turbulent flow
chromatography mean that non-digested, high molecular
weight proteins, as well as excess trypsin from the digestion
protocol, are excluded from interaction with column particles.
As such, these components do not reach the analytical HPLC
column, serving to increase column lifetime, and reduce the
freqency of MS ion-source maintenance – both important
considerations in high-throughput laboratories. TFA is not
favoured by some for MS analysis due to the risk of ion
suppression effects,[12] despite the chromatographic benefits
that can be gained for hydrophilic peptide analysis in
particular. In the method described, TFA was used to maximise
recovery on the TurboFlow columns, but was not transferred to
the analytical HPLC column or to the MS source guarding
against the possibility of ensuing ion suppression effects.
Using the focus-mode plumbing arrangement (Fig. 1), the
elution loop contents were diluted (1+9, v/v) prior to the
analytical HPLC column, such that the retained peptides
were focused at the head of the analytical column prior to
gradient elution. This focusing meant that no evaporation/
reconstitution steps were employed, thus avoiding the risk
of loss of some peptides during these steps.
Overall, the data from this study suggest that turbulent
flow chromatography offers a rapid, on-line alternative to
SPE for the analysis of peptide digests by LC/MS. A wide
range of TurboFlow column chemistries are available, and
so the technique can be further cutsomised for analyses
which are targeted to specific peptides. As with turbulent
flow chromatography for small-molecule workflows,[9] this
approach may be ideally suited to high-throughput
protein-based applications, such as those which are emerging
in clinical laboratories.
REFERENCES
In this study using a single protein and with the chosen
combination of TurboFlow column chemistries, good recovery
was observed for a number of the tryptic peptides (Table 3).
Recovery for the more hydrophilic BSA peptides was greater
using the two columns in series than for each of the columns
individually (data not shown), suggesting that both cation-
exchange and hydrophobic/hydrophilic interactions were
contributing to peptide recovery (analagous to mixed-mode
SPE methods). As one would predict, the relative recovery of
the more hydrophobic peptides was greater than for the
hydrophilic peptides. Recovery of the most hydrophilic
peptide, ATEEQLK, was relatively poor (15%), but nonetheless
showed good injection-to-injection peak area precision. One of
the 18 peptides in this study (VLASSAR) was not observed.
This was not considered to be due to poor recovery on the
TurboFlow column, since this peptide was not observed in LX
injections. It is more likely that this peptide was either not
produced, or was unstable during the digestion protocol.
In cases where a number of possible tryptic peptides can be
used, the choice of which surrogate peptide(s) to use for
quantitative protein analysis remains important,[13] and the
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