Mass spectrometry analysis of 2-nitrophenylhydrazine carboxy derivatized peptides

Article abstract of DOI:10.1007/s13361-011-0220-y

Peptides with two or more basic residues, including those with post-translational modifications (PTMs), such as methylation and phosphorylation, can be highly hydrophilic and, therefore, are often difficult to be retained on a reversed-phase (RP) column. In addition, these highly hydrophilic peptides may carry two or more positive charges, which often fragment poorly upon collisionally activated dissociation (CAD), resulting in few sequence-specific ions. C-terminal rearrangement may also occur during CAD. Furthermore, some PTMs are labile and tend to be lost when subjected to CAD as is the case with phosphorylation on serine or threonine. To overcome the difficulties of separation, detection, and fragmentation of highly hydrophilic peptides, we report here the effect of carboxy group derivatization with 2-nitrophenylhydrazine (this strategy will be called NPHylation for simplicity). NPHylation significantly increases the hydrophobicity of the peptides, eliminates C-terminal rearrangement in all cases, and offers enhanced sensitivity in some cases. In addition, the CAD spectra of the resulting NPHylated peptides carry more sequence-specific ions due to significant reduction of sequence scrambling as observed for peptide EHAGVISVL. Furthermore, the different carboxy derivatives of this peptide undergo sequence scrambling to varying degrees, which clearly demonstrates that the C-terminus has a profound effect on peptide fragmentation. Finally, sequence scrambling is a charge dependent phenomenon, which affects CAD of doubly charged peptides far more than their singly charged counterparts.

Full text of DOI:10.1007/s13361-011-0220-y

B American Society for Mass Spectrometry, 2011
J. Am. Soc. Mass Spectrom. (2011) 22:1958Y1967
DOI: 10.1007/s13361-011-0220-y
RESEARCH ARTICLE
Mass Spectrometry Analysis
of 2-Nitrophenylhydrazine Carboxy Derivatized
Peptides
Junmei Zhang, Rowaida Al-Eryani, Haydn L. Ball
Protein Chemistry Technology Center, Department of Internal Medicine, University of Texas Southwestern Medical Center,
Dallas, TX 75390-8816, USA
Abstract
Peptides with two or more basic residues, including those with post-translational modifications
(PTMs), such as methylation and phosphorylation, can be highly hydrophilic and, therefore, are
often difficult to be retained on a reversed-phase (RP) column. In addition, these highly
hydrophilic peptides may carry two or more positive charges, which often fragment poorly upon
collisionally activated dissociation (CAD), resulting in few sequence-specific ions. C-terminal
rearrangement may also occur during CAD. Furthermore, some PTMs are labile and tend to be
lost when subjected to CAD as is the case with phosphorylation on serine or threonine. To
overcome the difficulties of separation, detection, and fragmentation of highly hydrophilic
peptides, we report here the effect of carboxy group derivatization with 2-nitrophenylhydrazine
(this strategy will be called NPHylation for simplicity). NPHylation significantly increases the
hydrophobicity of the peptides, eliminates C-terminal rearrangement in all cases, and offers
enhanced sensitivity in some cases. In addition, the CAD spectra of the resulting NPHylated
peptides carry more sequence-specific ions due to significant reduction of sequence scrambling
as observed for peptide EHAGVISVL. Furthermore, the different carboxy derivatives of this
peptide undergo sequence scrambling to varying degrees, which clearly demonstrates that the
C-terminus has a profound effect on peptide fragmentation. Finally, sequence scrambling is a
charge dependent phenomenon, which affects CAD of doubly charged peptides far more than
their singly charged counterparts.
Key words: Peptide hydrophobicity, Hydrophilic peptide, Peptide carboxy group derivatization by
NPHylation, C-terminal elimination, Sequence scrambling, Fragmentation pattern, Collisionally
activated dissociation (CAD)
due to one or more missed cleavages. The extent of missed
cleavages increases when lysine or arginine carry post-
Introduction
rypsin is one of the most commonly used enzymes for
bottom-up proteomics. After a typical trypsin digestion,
some peptides will have two or more basic groups, partially
translational modifications (PTMs) such as lysine acetylation
and/or lysine/arginine methylation. Peptides with two or
more charged groups tend to be highly hydrophilic and are
not retained well on a reversed-phase (RP) chromatography
column. Phosphorylation is another example of a PTM that
increases the hydrophilicity of the peptide on which it is
attached. In addition to their very short retention times (if
retained at all), these peptides often have limited proton
mobility (due to strong sequestration of the ionizing proton
T
Electronic supplementary material The online version of this article
(doi:10.1007/s13361-011-0220-y) contains supplementary material, which
is available to authorized users.
Correspondence to: Haydn L. Ball; e-mail: Haydn.Ball@utsouthwestern.edu
Received: 21 March 2011
Revised: 14 July 2011
Accepted: 19 July 2011
Published online: 4 August 2011

J. Zhang, et al.: 2-Nitrophenylhydrazine for Peptide Carboxy Group Derivatization
1959
(s) by basic amino acid side chains) and undergo reduced
fragmentation along the peptide backbone upon CAD (in
agreement with the mobile proton model introduced by
Gaskell and Wysocki et al. [1, 2]), a commonly used low
energy fragmentation technique for proteomics studies.
PTMs, including lysine acetylation [3–7], lysine/arginine
methylation [8–11], and phosphorylation [12], are among
the most widespread modifications and they regulate many
cellular processes including cell survival and apoptosis,
cellular differentiation, metabolism, and signal transduction.
With increasing interest in such modifications and the
increasing use of mass spectrometry for analysis of PTMs,
efforts have been made to augment the hydrophobicity [13]
and retention time [13, 14] of these highly hydrophilic
peptides, or to apply an alternative fragmentation technique,
e.g., electron transfer dissociation (ETD), for better mapping
the PTMs like lysine/arginine methylation [14]. Since ETD
has only been implemented on a few instruments to date and
is not as widely available as CAD, we decided to evaluate
other molecules better suited for ESI-MS/MS experiments,
than reagents such as 1-(2-pyrimidyl)piperazine (PP) that
have been used in MALDI-TOF peptide mass fingerprint
applications [15].
using optimized Fmoc chemistry [16]. Crude peptides were
purified on a Waters 600 HPLC system (Milford, MA, USA)
using a Vydac C18 semi-preparative column (250 mm×
10 mm) at 3 mL/min and 0%–100% B in 120 min, where A
is water/0.045% TFA and B is acetonitrile/0.036% TFA. The
purified peptides were characterized using either MALDI-MS
or ESI-MS.
NPHylation of Peptides
Each of the six derivatization reagents (PP, 2-NPH, 3-NPH.
HCl, 4-NPH, 2,6-DMP, and 2-NAL) was allowed to react
with peptides according to a procedure reported previously
[13]. For NPHylation with 2-NPH, the reagent of particular
interest, the concentrations of 2-NPH, EDC, HOAt, and TFA
were varied during optimization of the reaction conditions.
The final conditions were as follows: 1.2 to 12.0 μL of 0.5%
2-NPH in DMF, 4 μL of 2 mg/mL EDC in DMF, and 3 μL
of 2 mg/mL HOAt were sequentially added to a solution of
peptide (G0.1 μg/μL) in 50 μL water. After vortexing for
30 s, the reaction mixture was dried in a SpeedVac and
desalted on C18 ZipTips (Millipore, Bedford, MA, USA)
prior to LC-MS/MS analysis.
In our studies a significant neutral loss was observed for
the PP derivatized threonine phosphorylated peptide
TPpTAPSLG upon CAD, which complicated MS/MS
spectrum interpretation. Thus, a total of five alternative
reagents were identified and tested, resulting in 2-nitro-
phenylhydrazine (2-NPH) becoming the molecule of choice.
The 2-NPH derivatized peptides have higher hydrophobicity
and longer retention time on a C18 column, exhibit
considerably less neutral loss compared with their PP
derivatized counterparts [13] in all cases, and have higher
sensitivity in some cases. NPHylation (derivatization with 2-
NPH) was also useful for understanding the fragmentation
mechanisms of peptides.
Nano-LC-MS/MS Analysis
Each peptide was reconstituted in 5–30 μL of HPLC
buffer A [0.1% formic acid/2% acetonitrile/97.9% H2O
(vol/vol/vol)] and 1 μL was injected into the Agilent 1100
nano flow HPLC system (Palo Alto, CA, USA). Mass
analysis was performed on a ThermoFisher LTQ – 2D ion
trap mass spectrometer (San Jose, CA, USA) equipped
with a nano-ESI source. The capillary column (10 cm
length×75 μm i.d.) was home packed with Luna C18 resin
(5 μm particle size, 100Å pore diameter) (Phenomenex,
Torrance, CA, USA). Peptides were eluted from the
column using a gradient from 20% to 90% buffer B
[0.1% formic acid/90% acetonitrile/9.9% H2O (vol/vol/
vol)] with a 40 min gradient. The eluted peptides were
directly electro-sprayed into the LTQ spectrometer with
MS/MS spectra acquired in a data dependent mode that
cycled between MS and MS/MS of the eight strongest
precursor ions. All the MS/MS experiments were per-
formed with 32% normalized collision energy.
Experimental
Materials and Chemicals
2-Nitrophenylhydrazine (98%, 2-NPH), 2,6-dimethylpiperi-
dine (98%, 2,6-DMP), and 2-nitroaniline (98%, 2-NAL) were
purchased from Alfa Aesar (Ward Hill, MA, USA). 1-(2-
Pyrimidyl)piperazine (98%, PP), 3-nitrophenylhydrazine
hydrochloride (98%, 3-NPH.HCl), 4-nitrophenylhydrazine
(97%, 4-NPH), and N-(3-dimethylaminopropyl)-N'- ethylcar-
bodiimide hydrochloride (EDC) were obtained from Sigma-
Aldrich (St. Louis, MO, USA). 1-Hydroxy-7-azabenzotriazole
(99.9%, HOAt) was purchased from GenScript (Piscataway,
NJ, USA).
Results and Discussion
It has been reported that derivatization of carboxy groups
with 1-(2-pyrimidyl)piperazine (PP) could improve the
hydrophobicity/gas phase basicity and increase the ioniza-
tion efficiency of phosphopeptides [13]. However, the effect
this derivatization has on the fragmentation pattern of such
peptides is unknown. Therefore, initial CAD studies were
performed using both phosphorylated and underivatized
peptides, comparing this data with that obtained using five
Peptide Synthesis
Peptides were synthesized on an Applied Biosystems 433
automated peptide synthesizer (Foster City, CA, USA),

1960
J. Zhang, et al.: 2-Nitrophenylhydrazine for Peptide Carboxy Group Derivatization
other reagents. The results indicated that 2-NPH was the best
for LC-MS/MS type experiments.
485.2 (doubly charged, minor component) with retention
time of 13.2 and 14.6 min, respectively, were observed for
the model peptide (Figure 2a). Mainly one group of such
ions with the longer RT was observed at higher intensities
under the conditions optimized for 2-NPH. Significant
neutral loss of 164Da occurred along with loss of the
phosphate group upon CAD of the molecular ions (Figure 3a
and b), which does not help with spectrum interpretation.
Please note that only CAD spectra of the most abundant ions
at m/z 969.4 are shown for simplicity. The CAD spectra of
Optimization of the Derivatization Reactions
Each of the six reagents (see Figure 1 for their structures)
was allowed to react with a model threonine phosphorylated
peptide TPpTAPSLG under the same experimental condi-
tions described by Xu et al. [13]. Two groups of the PP
derivatized molecular ions at m/z 969.4 (singly charged) and
Figure 1. Structures of the derivatization reagents tested in this work

J. Zhang, et al.: 2-Nitrophenylhydrazine for Peptide Carboxy Group Derivatization
1961
100
(a)
(b)
(c)
(d)
RT=11.7 min, m/z=823.4 (1+), 412.2 (2+)
RT=13.2 min, m/z=969.4 (1+), 485.2 (2+)
50
RT=14.6 min, m/z=969.4 (1+), 485.2 (2+)
0
100
RT=18.2 min, m/z=958.4 (1+), 479.7 (2+)
50
0
100
RT=12.6 min, m/z=823.4 (1+), 412.2 (2+)
RT=19.1 min, m/z=958.4 (1+), 479.7 (2+)
50
0
100
RT=18.6 min, m/z=958.4 (1+), 479.7 (2+)
RT=12.2 min, m/z=823.4 (1+), 412.2 (2+)
50
0
0
2
4
6
8
10
12
14
16
18
20
22
24
Time (min)
Figure 2. Total ion chromatograms of threonine phosphorylated peptide TPpTAPSLG after derivatization with the following
reagents: (a) 1-(2-pyrimidyl)piperazine (PP), (b) 2-nitrophenylhydrazine (2-NPH), (c) 3-nitrophenylhydrazine hydrochloride (3-
NPH.HCl), and (d) 4-nitrophenylhydrazine (4-NPH)
(b)
-164 -98
707.4
Δ
y4
519.4
100
50
0
100
50
0
(a)
-164
805.4
b7 -98
650.4
y6 -98
673.4
730.4
Δ
787.5
Δ
Δ
-98
b7
748.4
787.5
y6
771.4
-164
805.4
689.5
871.5
Δ
y6 -164 -98
632.4
y5
509.4
-164 -98
707.5
Δ
-b 8
951.4
590.4
434.3
300
500
700
m/z
900
1100
300
500
700
m/z
900
1100
Δ
y4
508.4
-153 -98
707.5
y4
(e)
508.4
(d)
Δ
100
50
0
100
50
0
100
50
0
(c)
y4
508.4
689.5
689.5
y6 -98
662.4
-153 -98
707.5
Δ
b7 -98
650.4
Δ
730.4
y6 -98
662.4
b7
748.4
b7 -98
650.4
632.5
Δ
-98
860.4
y6 -98
662.4
Δ
Δ
730.3
b4
451.3
b7 -98
650.5
y5
579.4
Δ
730.3
632.5
-98
860.4
-153
805.4
-98
335.3
Δ
860.4
y5
b4 -98
353.4
b4 -98
353.4
y5
579.4
632.4
b4
451.3
Δ
b7
748.3
Δ
Δ
b7
748.4
579.4
b4
451.3
940.3
940.3
940.3
∗
∗
300
500
700
m/z
900
1100
300
500
700
m/z
900
1100
300
500
700
m/z
900
1100
Figure 3. CAD spectra of singly charged peptide TPpTAPSLG after derivatization with the following reagents: (a) and (b) 1-(2-
pyrimidyl)piperazine (PP) with RT 13.2 min (a) and 14.6 min (b), respectively, (c) 2-nitrophenylhydrazine (2-NPH), (d) 3-
nitrophenylhydrazine hydrochloride (3-NPH.HCl), and (e) 4-nitrophenylhydrazine (4-NPH). The open triangle (Δ) designates “b”,
“y” or “a” ions with water and/or ammonia loss. The asterisk (*), when present, designates the precursor ion. The same
nomenclature is used for all subsequent figures

1962
J. Zhang, et al.: 2-Nitrophenylhydrazine for Peptide Carboxy Group Derivatization
the corresponding doubly charged ions at m/z 485.2 are
reported in the Supplemental Information section (SF1). The
other PP derivatized peptides also have more neutral loss of
the derivatization reagent (PP: 164 Da in this case) than the
2-NPH derivatized counterparts (2-NPH: 153 Da)
(Figure 3c). Thus, derivatization of peptides with PP may
significantly boost the ionization efficiency of the phospho-
peptides for MALDI peptide mass fingerprint experiments,
yet it is not a method of choice if CAD has to be used for
identification of the peptides when only a low resolution
instrument is available.
Since the first reports in the early 1990s [17–20], 2-NPH
and 2-NPH.HCl (2-nitrophenylhydrazine hydrochloride)
have been used to derivatize fatty acids and other carboxylic
acids, aldehydes, and ketones, for improved retention
properties on a RP column and for more sensitive UV
detection. Based on the carboxy group reactivity of 2-NPH
and 2-NPH.HCl and their structurally similar homologs, we
investigated whether or not derivatization could facilitate the
identification of peptides by MS/MS by generating more
sequence-specific fragment ions.
desired derivatization products but 2-NPH, 3-NPH.HCl, and
4-NPH converted the test peptides to their NPHylated
products (peptide +135 Da), which have a longer RT than
both their underivatized and PP-derivatized counterparts
(Figure 2). While the RT of the three NPHylation products
was similar, the NPHylation reaction seemed to be more
complete when 2-NPH was used (Figure 2b versus Figure 2c
or d). More importantly, the extent of neutral loss of 153 Da
(the NPHylation reagent) was the least significant when the
peptides were NPHylated with 2-NPH (Figure 3 and SF1).
When a serine phosphorylated peptide TPTAPpSLG was
NPHylated with 2-NPH, its CAD spectra also showed very
minimal loss of 153 Da (SF2). In addition to the phosphopep-
tides, three tryptic peptides were tested: AYPGFK, GRID-
KPILK and ISQAVHAAHAEINEAGR, and similar trends of
neutral loss of 153 Da were observed. Therefore, 2-NPH was
chosen as the reagent for all subsequent experiments.
Selectivity and Derivatization Efficiency of 2-NPH
For peptides that do not contain aspartic or glutamic acid, 2-
NPH reacts rapidly with the C-terminus of the peptides. One
such example is shown in Figure 4a for N-terminal
Among the five reagents tested, 2,6-dimethylpiperidine
(2,6-DMP) and 2-nitroaniline (2-NAL) did not produce the
(a)
100
7.7 min, M + 135
2.2 min, M + 135
#
0
100 (b)
6.1 min, M + 135 + 135
2.1 min, M + 135 +135
#
4.6 min, M + 135
#
#
0
100
(c)
18.6 min, M + 135 -153
14.9 min, M + 135
10.1 min, M
#
#
#
#
#
0
100
(d)
18.5 min, M + 135
19.9 min, M + 135 -18
#
18.9 min, M + 135 -153
#
0
0
2
4
6
8
10
12
14
16
18
20
22
24
Time (min)
Figure 4. Selectivity of peptides for derivatization with 2-nitrophenylhydrazine (2-NPH): (a) Ac-KKKANLQYYA, (b)
VHGQPHQDTK, (c) SIINFEKL, and (d) EYIPTVF. For simplicity, the peptides are labeled with M and their NPHylation products
are labeled with M +135, and M +135 +135. Neutral loss of 2-NPH and water are labeled with −153 and −18, respectively.
Peaks irrelevant to the peptides of interest are labeled with the number symbol (#)

J. Zhang, et al.: 2-Nitrophenylhydrazine for Peptide Carboxy Group Derivatization
1963
acetylated peptide Ac-KKKANLQYYA. When an aspartic
and/or glutamic acid residue exists in a peptide, 2-NPH
predominately reacts with the C-terminus, yet it does show
some reactivity towards carboxy side chain moiety of these
amino acids, though the extent of such derivatization varies
with the identity and location of these groups. For example,
the major product of peptide VHGQPHQDTK is addition of
2*135 Da (Figure 4b), while that of SIINFEKL is addition
of 1*135 Da (Figure 4c), with a fragmentation pattern that
agrees with derivatization of the C-terminus (SF3). It should
be pointed out that due to the impurities in our synthetic
peptide preparations, there are some peaks present that are
not related to the peptides of interest, and they are labeled
with the number symbol (#).
1:1 ratio before injection and eluted with 1.2 μL/min
90% B (to reduce the extent of peak broadening for a
more objective evaluation of sensitivity). The ratio of ion
intensity/peak height of the NPHylation products versus
underivatized peptides was 1.2, 1.4, 15.7, 12.5, and the
peak area ratio was 1.4, 1.5, 6.3, and 2.5, respectively
(Figure 5). Thus, the sensitivities vary from increasing
slightly or up to several fold upon NPHylation. Though
it is not ideally affecting sensitivity to the same extent,
NPHylation does offer better detection limits for some
hydrophilic peptides. More importantly NPHylation provides
better retention of hydrophilic peptides, increased hydro-
phobicity, and significant reduction of non sequence-specific
fragment ions as described below.
Except for SIINFEKL, the other three peptides were
almost completely converted to their NPHylation products
after 2-NPH derivatization (Figure 4). It is rather difficult to
estimate the reaction yield since the peptides and their
NPHylated products have different RT, peak shapes, and
sensitivities. Our best estimation is that the efficiency of
NPHylation reaction is about 80% or higher. Besides the
desired NPHylation derivatives, loss of 2-NPH from the
derivatives has been sometimes observed as a side product
(Figure 4c and d). In addition, loss of water from the
NPHylated peptides was observed as a minor product when
glutamic acid is the N-terminal residue such as EYIPTVF, in
agreement with the literature [21–23] (Figure 4d).
NPHylation Reduces Non-Sequence-Specific
Fragment Ions
Two major sources of non-sequence-specific fragment ions
are C-terminal elimination and sequence scrambling, which
can be blocked or reduced by NPHylation, respectively.
C-terminal Elimination (C-terminal Rearrangement) Hydro-
philic peptides with two or more basic groups have a
tendency to undergo C-terminal elimination (loss of the
C-terminal residue) upon CAD, a phenomenon already
documented in the literature [24–36] and recently found
to be a charge-dependent, side-chain assisted C-terminal
rearrangement [29]. By blocking the C-terminal carboxy
group, NPHylation completely eliminates such C-terminal
rearrangement for both charge states (SF4), once again
demonstrating that a free C-terminal carboxy group is
required for C-terminal elimination side reactions [26,
29, 36]. Therefore, NPHylation not only improves the
hydrophobicity of hydrophilic peptides but also elimi-
nates an undesirable C-terminal rearrangement, making
NPHylation an attractive alternative for tandem mass
spectrometry studies.
NPHylation Increases Peptide Hydrophobicity
NPHylation with 2-NPH significantly improves the hydro-
phobicity of the peptides and increases their retention time
on a RP column. For example, the RT of threonine
phosphorylated peptide TPpTAPSLG increased from
~11.5 min to ~18.2 min after NPHylation with 2-NPH
(Figure 2b), which is longer than the equivalent PP
derivatized analogs, that eluted at ~13.2 or 14.6 min
(Figure 2a). The RT of the underivatized versus NPHylated
peptides listed in Figure 4 follows the same trend: 5.9 versus
7.7 min for peptide Ac-KKKANLQYYA; 2.1 versus 4.6 min
(mono-NPHylated) or 6.1 min (di-NPHylated sequence) for
VHGQPHQDTK; 9.8 versus 14.9 min for SIINFEKL; and
11.6 versus 18.5 min for EYIPTVF. Please note that
Ac-KKKANLQYYA and VHGQPHQDTK are highly
hydrophilic peptides that are difficult to be retained on a
RP column, especially VHGQPHQDTK. Thus, NPHylation
facilitates the detection of these highly hydrophilic peptides
that would otherwise be lost in the void volume, as a result
of the increased hydrophobicity imparted by this chemical
modification.
Sequence Scrambling In addition to C-terminal rearrange-
ment, sequence scrambling is another phenomenon that
produces non-sequence-specific fragment ions, and thus
limits our abilities for correct peptide identification [37–
58]. It has been found that N-terminal acetylation eliminates
sequence scrambling [37, 41, 55], as well as the type of
amino acid side-chains (not including the C-terminus), i.e.,
acidic, basic, or amide, and their positions apparently
influence the extent of sequence scrambling [46, 47, 53,
55]. We investigated the effect of NPHylation on this
phenomenon at the MS/MS level.
Taking peptide EHAGVISVL as an example, several
sequence scrambling ions can be seen from the CAD
spectrum of the doubly charged precursor ion: b08 À I (m/z
662.3, relative abundance 31%), b8 – I (m/z 680.3, 7%), and
b08 À IV (m/z 563.4, 1%), which is in agreement with the
NPHylation Increases the Peptide Sensitivity
in Some Cases
To quickly assess the sensitivity of NPHylation, model
peptides and their NPHylated counterparts were mixed at

1964
J. Zhang, et al.: 2-Nitrophenylhydrazine for Peptide Carboxy Group Derivatization
100
(a)
M + 135
M
0
100
(b)
(c)
(d)
M + 135
M
0
100
M + 135 + 135
M, M + 135, M + 135 + 135
M + 135
0
100
M + 135
M
0
0
1
2
3
4
5
6
7
8
9
10
Time (min)
Figure 5. Sensitivity of peptides for derivatization with 2-nitrophenylhydrazine (2-NPH): (a) TPpTAPSLG, (b) Ac-KKKANLQYYA, (c)
VHGQPHQDTK, and (d) K(Ac)SAPATGGVK. They were eluted with 90% B at 1.2 μL/min for easy comparison of sensitivity
literature [41]. After NPHylation, the relative abundances of
the non-sequence-specific ions are decreased over 6-fold to
5%, 1%, and G1% (not visible), respectively (Figure 6b),
significant enough to suggest that NPHylation can reduce
the extent of sequence scrambling at the MS/MS level.
Similar reduction of sequence scrambling was also observed
for 4-NPH and 3-NPH.HCl derivatized counterparts of the
same peptide (SF5).
In addition to the above three NPH reagents, PP
derivatization reduced the extent of sequence scrambling
such that no obvious scrambling ions are detected
(Figure 6c). To demonstrate that such an effect is not solely
due to the bulkiness of the modified C-terminus, the C-
terminally amidated version of the peptide (EHAGVISVL-
NH2) was synthesized and subjected to CAD. Interestingly,
loss of ammonia became the dominant fragmentation path-
way as observed before [42], and no obvious sequence
scrambling was observed at the MS/MS level (Figure 6d),
clearly indicating that factors other than steric hindrance of
the C-terminus affect the fragmentation of protonated
peptides.
derivatives (2-NPH, 3-NPH.HCl, and 4-NPH) undergo
sequence scrambling at the +2 charge state though to a
different extent (Figure 6a and b, SF5), however, their
singly charged counterparts do not seem to exhibit this
same phenomenon (SF6). Since peptide identification
relies heavily on CAD spectra of doubly charged ions
and doubly charged ions are more likely to undergo
sequence scrambling than their singly charged counter-
parts, our efforts to reduce the extent of (if not eliminate)
sequence scrambling and understand its underlying
mechanism(s) are well rationalized. Sequence scrambling
is believed to involve formation of a cyclic intermediate
produced by the nucleophilic attack of the N-terminal
nitrogen over an electrophilic carbonyl carbon near the
C-terminus and its subsequent reopening at different
positions [39, 42]. It is beyond the scope of this work
to study the mechanisms of sequence scrambling, but we
can see that the phenomenon becomes less favored at the
MS/MS level when the protons become sequestered/less
mobile by a nucleophilic modification, such as NPHya-
tion or amidation of the C-terminus. Anecdotally, it has
been shown by Harrison and his co-workers that histidine
containing b ions undergo sequence scrambling, which is
observable at the MS/MS/MS level [53]. Thus, care is
needed when comparing sequence scrambling studies at
Moreover, sequence scrambling is more significant for
doubly charged ions (C-terminal rearrangement is gen-
erally observable with singly charged ions). For example,
peptide EHAGVISVL and each of its three NPH

J. Zhang, et al.: 2-Nitrophenylhydrazine for Peptide Carboxy Group Derivatization
1965
b8
793.4
y7/b8
793.4
100
100
(a)
(b)
y3/(-153) 2+
453.9
Δ
y4
431.3
b08 -I
662.3
b7
694.4
388.2
a2+
b6
607.4
8
b2+
Δ
Δ
8
b5
383.5
Δ
b8 -I
680.3
397.3 453.9
589.3
494.3
Δ
Δ
589.3
b6
607.4
444.8
Δ
y4
374.5
Δ
50
b08 -IV
50
521.3
566.3
Δ
775.3
y1/b2
563.4 b08 -I
775.3
Δ
+
267.2
SV + H
187.1
662.3
Δ
b2+
757.4
b5
8
y3
757.3
Δ
397.5
494.2
b7
318.2
250.2
Δ
Δ
y2
y8
795.4
694.4
676.4
388.5
231.2
0
0
200
400
600
m/z
800
200
400
600
m/z
800
Δ
Δ
(c)
(d)
100
100
527.1
453.8
50
50
y1
278.3
Δ
888.4
(-46) 2+
439.9
Δ
+
(PP + H)
166.2
Δ
-164
906.5
444.9
775.4
b8
793.4
(-164) 2+
453.9
Δ
b8
793.4
b2+
397.2
775.4
b6
607.3
8
0
0
200
400
600
m/z
800
1000
200
400
600
800
m/z
Figure 6. CAD spectra of doubly charged peptide EHAGVISVL: (a) without derivatization, (b) after derivatization with
2-nitrophenylhydrazine (2-NPH), (c) 1-(2-pyrimidyl) piperazine (PP), and (d) amidation of the C-terminus
different stages of tandem mass spectrometry. As a result
of the reduction in proton mobility, the singly charged
peptides undergo sequence scrambling to a lesser extent.
Although it is not our initial intention to study in detail
the mechanism of sequence scrambling or neutral loss,
our data suggest that altering the proton availability and/
or mobility of a peptide by derivatizing its C-terminus
and/or changing its charge state can have a profound
effect on peptide fragmentation. That is to say, the C-
terminus can play an important role in the process of
sequence scrambling.
It is interesting to point out that scientists have started
to study the effect of sequence scrambling on a proteo-
mics scale [57, 59, 60], which inevitably involves a
database search engine, such as Mascot. The current
version of Mascot 2.3 is “not trying to find all possible
matches in a spectrum” (personal communications with
technical support) such that similar scores can be obtained
for a peptide with and without considering sequence
scrambling ions. At least partially due to the way Mascot
scores, our NPHylation strategy does not give much
improvement in peptide scoring even though manual
inspection gives a different picture. Though it is still
debatable, we believe more sophisticated software that
better handles neutral losses will benefit the proteomics
community and strategies like NPHylation will become
more attractive.
Conclusion
For successful bottom-up proteomics analysis, effective
separation on a RP column, sensitive detection, and
sequence-specific fragmentation of peptides are required.
Hydrophilic peptides with multiple charged groups or
carrying PTMs can easily elude detection and identification
since they are not readily retained on the column, and may
produce non-sequence-specific fragment ions. Furthermore,
negatively charged PTMs such as phosphorylation and
sulfation may suppress ionization in the mass spectrometer
resulting in reduced sensitivity. To address these issues, 2-
NPH was used as a derivatization reagent to react with the
carboxy group(s) of the peptides, with the C-terminus as the
preferred site of reaction. NPHylation significantly improves
the hydrophobicity, increases the retention times in all cases,
and offers higher sensitivity for some peptides. Moreover,
NPHylation eliminates C-terminal rearrangement reactions
and reduces the extent of sequence scrambling, thereby
generating more sequence-specific fragment ions. By com-
paring the fragmentation patterns of the original and
NPHylated peptide pairs, insights into the mechanism of

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