Proteomics 2009 V9p1683

Proteomics 2009, 9, 1683–1695 DOI 10.1002/pmic.200800562 1683

RESEARCH ARTICLE

The detection, correlation, and comparison of
peptide precursor and product ions from data
independent LC-MS with data dependant LC-MS/MS
Scott J. Geromanos1, Johannes P. C. Vissers2, Jeffrey C. Silva1*, Craig A. Dorschel1,
Guo-Zhong Li1, Marc V. Gorenstein1, Robert H. Bateman2 and James I. Langridge2
1
Waters Corporation, Milford, MA, USA
2
Waters Corporation, Manchester, UK

The detection, correlation, and comparison of peptide and product ions from a data independent LC- Received: July 3, 2008
MS acquisition strategy with data dependent LC-MS/MS is described. The data independent mode of Revised: September 18, 2008
acquisition differs from an LC-MS/MS data acquisition since no ion transmission window is applied Accepted: October 1, 2008
with the first mass analyzer prior to collision induced disassociation. Alternating the energy applied to
the collision cell, between low and elevated energy, on a scan-to-scan basis, provides accurate mass
precursor and associated product ion spectra from every ion above the LOD of the mass spectrometer.
The method therefore provides a near 100% duty cycle, with an inherent increase in signal intensity
due to the fact that both precursor and product ion data are collected on all isotopes of every charge-
state across the entire chromatographic peak width. The correlation of product to precursor ions, after
deconvolution, is achieved by using reconstructed retention time apices and chromatographic peak
shapes. Presented are the results from the comparison of a simple four protein mixture, in the pres-
ence and absence of an enzymatically digested protein extract from Escherichia coli. The samples were
run in triplicate by both data dependant analysis (DDA) LC-MS/MS and data-independent, alternate
scanning LC-MS. The detection and identification of precursor and product ions from the combined
DDA search results of the four protein mixture were used for comparison to all other data. Each in-
dividual set of data-independent LC-MS data provides a more comprehensive set of detected ions than
the combined peptide identifications from the DDA LC-MS/MS experiments. In the presence of the
complex E. coli background, over 90% of the monoisotopic masses from the combined LC-MS/MS
identifications were detected at the appropriate retention time. Moreover, the fragmentation pattern
and number of associated elevated energy product ions in each replicate experiment was found to be
very similar to the DDA identifications. In the case of the corresponding individual DDA LC-MS/MS
experiment, 43% of the possible detectable peptides of interest were identified. The presented data
illustrates that the time-aligned data from data-independent alternate scanning LC-MS experiments
is highly comparable to the data obtained via DDA. The obtained information can therefore be effec-
tively and correctly deconvolved to correlate product ions with parent precursor ions. The ability to
generate precursor-product ion tables from this information and subsequently identify the correct
parent precursor peptide will be illustrated in a companion manuscript.

Keywords:
Biomarker discovery / Data-independent LC-MS / Multiplexed LC-MS / Shotgun
sequencing / Time-resolved mass spectrometry

1 Introduction
Correspondence: Dr. Johannes P. C. Vissers, Waters Corporation,
Atlas Park, Simonsway, Manchester M22 5PP, UK MS has evolved into a powerful tool for the analysis of pro-
E-mail: hans_vissers@waters.com tein mixtures owing to its speed, sensitivity, and accuracy.
Fax: 144-161-435-4444

Abbreviations: BPI, base peak intensity; CID, collisional induced * Current address: Cell Signaling Technology, Inc., 3 Trask Lane,
dissociation; DDA, data dependant analysis Danvers, MA 01923, USA

© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

1684 S. J. Geromanos et al. Proteomics 2009, 9, 1683–1695

The technology has played a pivotal role in the postgenomic increasing sample complexity. This creates a fundamental
era, helping to define the functional roles of identified gene problem for the identification of proteins over a wide dy-
products and gain a deeper understanding of cellular biology. namic range, and/or the ability to generate any semblance of
Traditional mass spectrometric approaches for the identifi- protein sequence coverage. That is, in a DDA experiment, an
cation of peptides from enzymatically digested proteins individual precursor is subjected to MS/MS until the sum-
include MALDI-TOF MS for single proteins, or very simple med intensity of a given fragment ion reaches an acceptable
mixtures [1, 2], and LC-MS/MS for more complex mixtures number of ion counts. In the case of weak precursor ions,
[3–5]. As sample complexity increases in terms of the abso- this can result in using valuable time in the MS/MS mode of
lute number, dynamic range, and molecular weight of the acquisition trying to reach a threshold that cannot be
proteins present, the use of precursor mass measurements achieved. Furthermore, the allotted MS/MS acquisition time
alone, as utilized by MALDI-TOF PMF, does not provide must be limited in order to maximize the duty cycle of the
sufficient specificity to impart unambiguous protein identi- mass spectrometer. For example, in the instance of a 30 s
fication. In these instances, the samples are typically ana- wide chromatographic peak, and with an MS/MS acquisition
lyzed by an electrospray LC-MS/MS approach using a data speed of 200 ms, only approximately 1/150 of the peak vol-
dependant acquisition (DDA) method. The major advantage ume will be sampled. If the peptide of interest is present at
of this approach is the generation of primary structural the level of 1.5 fmol, and with a column flow rate of 300 nL/
information from the peptide precursor ion selected for min and an assumed ion transfer efficiency from the liquid
fragmentation. The added specificity provided by the frag- phase to the detector of 0.001 [7], the maximum amount
ment ion information increases the quality of peptide iden- available for detection is approximately 10 zmol. The ability
tifications from more complex protein mixtures. Despite to generate a good quality MS/MS fragment ion spectrum
being a more efficient strategy to identify proteins in com- and confident database search identification from such a
plex matrices, there are some inherent limitations associated limited amount is challenging.
with the technique. In order to obtain the highest possible sensitivity and by
These limitations become evident with the desire to taking the aforementioned into consideration, an MS1 ion
categorize and quantify proteins in increasingly complex transmission/isolation window around the precursor ion is
biological matrices. A detailed understanding of the changes typically set to 6 1.5–3 mass units, allowing for the selection
in the protein complement of these samples when they are of the complete isotopic distribution of the precursor ion of
stressed or compared to a perturbed biological system is interest with maximum sensitivity. This may not be a prob-
increasingly required. In order to reach this goal, there are a lem for the more abundant precursor ions or peptides that
number of bioanalytical challenges that must be understood were selected for an MS/MS acquisition near their chroma-
and overcome to afford such comparative analyses. Firstly, tographic apex; however, the majority of the precursors are in
enzymatically derived peptides from proteins do not share lower intensity regime. In addition, more than one precursor
the same ionization efficiency. More specifically, they illus- is often present within the ion transmission window [8, 9],
trate an ionization distribution that spans close to two or resulting in product ion fragmentation components that do
three orders of magnitude, whereby the majority of the pep- not exclusively belong to the selected precursor. More speci-
tide signals are in the lower end of the distribution [6]. Sec- fically, Hoopmann et al. [8] found that in 17% of all MS/MS
ondly, the concentration of the proteins present in these events, more than one precursor was present in the collision
complex samples can, and often do, illustrate an even wider cell, whereby in a more recent study by Luethy et al. [9] this
dynamic range. Consequently, the majority of proteins pres- number was even found to be closer to 67% of all isolated
ent in a sample are at least two to three orders of magnitude precursors. Although this may not be an issue if there is a
lower in concentration than the most abundant protein. significant difference in precursor ion intensities and the
Hence, the majority of the tryptic peptides present in biolog- more intense species are of interest. It can however make a
ical matrices are in the lowest intensity regime. significant difference if the chimeric precursor ions present
The latter poses one of the main analytical challenges are of similar intensity or similar composition [9].
with respect to reproducibility in LC-MS/MS-based protein Many of these limitations described have a direct effect
identification schemes. A DDA experiment is typically a on the lack of reproducibility, low sequence coverage, and
serial process. The cycle starts by acquiring an MS survey large number of single peptide-based protein identifications
scan followed by the selection of a number of precursor ions present in the literature [10, 11]. As an example, in a pre-
for an MS/MS experiment that may or may not be at their viously published independent study, six proteomics labora-
chromatographic apex. The selected precursor ions are seri- tories analyzed a tryptic digest of complex biological matrix,
ally isolated for an MS/MS acquisition for an allotted period generated from 10 000 human cells by means of LC-MS/MS
of time, or until a certain ion current is breached. The num- [12]. In total 1757 proteins were identified of which only 52
ber of selected precursor ions and the allocated MS/MS proteins were commonly identified by all laboratories. In
acquisition time are optimized for a given sample type and another study [13], the results from three different multi-
complexity. Typically, the number of selected precursors will dimensional LC-MS strategies were compared with a fourth,
increase and the MS/MS acquisition time decrease with classical source, that is, protein biochemistry and clinical


Proteomics 2009, 9, 1683–1695 1685

chemistry literature. Combining the data sets of the various and label-free relative quantification on complex biological
approaches resulted in 1175 identified proteins. Interest- matrices [16] and has been applied for the characterization of
ingly, only 46 proteins were identified by all methods and Escherichia coli cultured using various carbon sources [24],
only 195 proteins by at least two methods. A third example the response of Mycobacterium bovis to isoniazid treatment
study involved the analysis of a glomerulus proteome [14]. [25], identifying, validating, and measuring the absolute
Here, the sample was prefractionated into 90 fractions using concentration of established markers in serum samples from
1D SDS-PAGE, and 2D solution phase IEF, in combination Gaucher patients [26] and exosomes secretion by oligoden-
with SDS–PAGE. Each fraction generated was analyzed by drocytes [27]. Here, it will be demonstrated that data inde-
LC-MS/MS. After validating the MS/MS data, it was deemed pendently acquired LC-MS data holds the same information
that 5% of these spectra could be processed for identification. in terms of detectable precursor and product ions compared
The combined search results identified 6686 proteins of to data dependent acquired data. Also shown is how pre-
which 45% were comprised of single peptide identifications. cursor and product ions are correlated within and across
A large number of tryptic peptides per protein and high experiments of various types and how this ultimately leads to
sequence coverage should however be expected using a frac- a 2–2.5-fold increase in detected components in complex
tionation strategy combined with the high sensitivity of an biological data sets.
LC-MS/MS approach.
The lack of reproducibility and the number of single
peptide identifications is however not primarily due to the 2 Materials and methods
experimental models employed, instrumentation and/or
software used to process and search the data, but to the 2.1 Sample preparation
method of data acquisition. To overcome some of these
problems, a data independent mode of acquisition was 50 mL of 0.5% aqueous formic acid was added to 100 mg of
introduced for label free quantitative LC-MS studies [15, 16], cytosolic E. coli digest standard (Waters, Milford, MA, USA).
whereby both precursor and product ion information is col- A tryptic digest stock solution containing four standard pro-
lected on all of the isotopes of all charge-states of the eluting teins, alcohol dehydrogenase, phosphorylase B, albumin,
peptide precursor ions across the chromatographic peak and enolase, was prepared in 0.1% aqueous formic acid and
width. Setting the MS acquisition speed in proportion to the diluted to concentrations of 200, 200, 200, and 100 fmol/mL,
chromatographic peak width ensures that a sufficient num- respectively. Equal volumes of the E. coli digest and the
ber of data points are collected from each precursor ion to standard proteins were combined to give a sample con-
adequately measure the m/z values, retention times, and centration of 0.5 mg/mL of E. coli digest and 100, 100, 100, and
peak volumes of all detectable ions. Other data-independent 50 fmol/mL of alcohol dehydrogenase, phosphorylase B,
methods have been reported, investigating the use of multi- albumin, and enolase, respectively. The tryptic digests of the
plexed fragmentation where more than one precursor ion four proteins were also prepared in 0.1% aqueous formic
was simultaneously fragmented by collisional induced dis- acid without the presence of the E. coli digest standard at the
sociation (CID) on Fourier transform ICR mass spectro- same concentration level of 100, 100, 100, and 50 fmol/mL,
meters [17, 18], IT mass spectrometers [19, 20], and TOF respectively. Unless stated otherwise, these solutions were
mass spectrometers [21]. Multiplexed or parallel CID, con- used as stocks for all the experiments described in this
ducted exclusively in the source region [18, 21], gas cell [22], manuscript.
or a combination of both [23] have been subject of research
papers too. All presented methods have in common that the 2.2 LC-MS configuration
detected product ions have to be associated to their parent
precursor. Hoaglund-Hyzer et al. [22] have suggested and Nanoscale LC separation of tryptic peptides was performed
applied the use of ion mobility gas phase separation to cor- with a nanoACQUITY system (Waters), equipped with a
relate simultaneously fragmented precursor ions to their Symmetry C18 5 mm, 5 mm6300 mm precolumn and an
concurrent product ions. The method applied in this study Atlantis C18 3 mm, 15 cm675 mm analytical RP column
employs liquid phase separation and time-alignment to cor- (Waters). The samples, 1 mL full loop injection, were initially
relate precursor ions with product ions. The m/z measure- transferred with an aqueous 0.1% formic acid solution to the
ments are converted to monoisotopic peptide masses, the precolumn at a flow rate of 4 mL/min for 3 min. Mobile
intensities of all isotopes and charge states summed, and the phase A was water with 0.1% formic acid whilst mobile
apex retention time for each species calculated. Next, the phase B was 0.1% formic acid in ACN. After desalting and
product ions are time-aligned and correlated to precursor preconcentration, the peptides were eluted from the pre-
ions whose apex retention time is within one-tenth of the column to the analytical column and separated with a gra-
time associated to each precursor ions chromatographic peak dient of 3–40% mobile phase B over 90 min at a flow rate of
width at half-height. 300 nL/min, followed by a 10 min rinse with 90% of mobile
The information obtained from the chromatographic re- phase B. The column was re-equilibrated at initial conditions
producibility of replicates can be used to accommodate label for 20 min. The column temperature was maintained at



357C. The lock mass compound, [Glu1]-fibrinopeptide B, was equalled 25 ppm and 0.05 Da, respectively, one missed
delivered by the auxiliary pump of the LC system at 250 nL/ cleavage site was allowed, and CAM-cysteine as a fixed and
min at a concentration of 100 fmol/mL to the reference methionine oxidation set as a variable modification. Addi-
sprayer of the NanoLockSpray source of the mass spectrom- tional protein identification reporting criteria included a
eter. All samples were analyzed in triplicate. peptide identification probability .95% and the presence of
Mass spectrometric analysis of tryptic peptides was per- a consecutive y ion series of at least three amino acids per
formed using a Q-Tof Premier mass spectrometer (Waters, MS/MS spectrum. A more comprehensive description of the
Manchester, UK). Accurate mass LC-MS data were collected DDA search algorithm has been described by Skilling et al.
in an alternating, low, and elevated energy mode of acquisi- [28].
tion (LC-MSE) [15, 16]. The spectral acquisition time in each Time-aligned LC-MSE precursor and product ions were
mode was 1.5 with an 0.1 s interscan delay. In low energy MS considered matched to a DDA identification provided that
mode, data were collected with a constant collision energy of the deconvoluted, protonated precursor ion mass, and
4 eV. In elevated energy MS mode, the collision energy was retention time were within 610 ppm and 630 s, respec-
ramped from 15 to 40 eV during each 1.5 s integration. One tively, and that there were a minimum of three product
cycle of low and elevated energy data were acquired every ions to match within 620 ppm. Additional data analysis
3.2 s. The RF applied to the quadrupole mass analyzer was were performed with Decision Site 9.0 (Spotfire, Somer-
adjusted such that ions from m/z 300 to 2000 were efficiently ville, MA USA) and Microsoft Excel (Microsoft, Redmond,
transmitted; ensuring that any ions observed in the LC-MS WA, USA).
data less than m/z 300 were known to arise from dissocia-
tions in the collision cell. For all measurements, the mass
spectrometer was operated in v-mode with a typical resolu- 3 Results and discussion
tion of at least 10 000 FWHM. All analyses were performed
in positive mode ESI. The TOF analyzer of the mass spec- 3.1 Alternate scanning data acquisition
trometer was externally calibrated with a NaI mixture from
m/z 50 to 1990. The data were postacquisition lock mass The alternate scanning acquisition method (LC-MSE) is
corrected using the doubly protonated monoisotopic ion of designed to collect high resolution, 10 000 v-mode or
[Glu1]-fibrinopeptide B. The reference sprayer was sampled 18 000–20 000 (FWHM) in the w-mode of instrument
every 30 s. operation, accurate mass information for each detected
Accurate mass LC-MS/MS DDA data were obtained as precursor and any corresponding fragment ion above the
follows. MS survey scans of 0.6 s duration with an interscan LOD of the mass spectrometer. The LC-MSE data acquisi-
delay of 0.05 s were acquired. MS/MS data were obtained for tion mode is configured to alternate between two collision
up to three ions of charge 21, 31 or 41 detected in the survey energy conditions. A low energy MS survey of eluting pre-
scan. MS/MS was obtained at a scan rate of 0.6 with 0.05 s cursor peptides and an elevated energy MS survey of asso-
interscan delay and a collision energy ramp from 15 to 40 eV. ciated product ions with no precursor ion selection applied
A dynamic exclusion window was set to 60 s. Acquisition was prior to CID. During the elevated energy MS survey, the
switched from MS to MS/MS mode when the base peak potential energy difference across the collision cell is
intensity (BPI) exceeded a threshold of 150 counts, and ramped in a linear fashion from an initial elevated energy
returned to the MS mode when the TIC in the MS/MS setting to a final value, over the period of time associated to
channel exceeded 1000 counts/s or when 0.9 s (three scans) a single scan. The LC-MSE data of a proteolytic digest are
were acquired. collected throughout the entire LC-MS experiment preser-
ving the chromatographic profile of all the detected peptides
2.3 Data processing and protein identification and their associated product ions. Product ion information
is obtained from all the isotopes and charge states of any
The continuum LC-MSE and DDA LC-MS/MS data were given precursor peptide as they are simultaneously frag-
processed using the default parameter settings for both mented. The principle of an LC-MSE acquisition has been
methods of acquisition as residing in ProteinLynx Global previously described in more detail [16].
SERVER version 2.3. The processed DDA data were queried The time-alignment correlation principle is illustrated in
against a Comprehensive Microbial Resource (http:// Fig. 1. It is based upon the principle that the chromato-
cmr.jcvi.org) E. coli K12 database (November 2007, 4403 graphic behavior of all ions associated to an eluting parent
entries) appended with a one-time randomized version of the peptide precursor is similar. Namely, product ions have the
database, the four spiked proteins and trypsin. The rando- same chromatographic profile as their parent precursor ions.
mized proteins sequences serve as a decoy to validate the Selecting the appropriate scan speed ensures that the chro-
identifications of the peptide. The DDA searches were con- matographic attributes of an ion can be accurately measured.
ducted with the default search engine parameters of the These attributes include peak area, accurate mass, retention-
software. Trypsin was set as the primary digest reagent, the time apex, peak width, and charge-state. Moreover, they pro-
precursor mass tolerance and fragment ion tolerances vide means for the elevated energy product ions to be time-


Proteomics 2009, 9, 1683–1695 1687

peak width. The width of the filter in the spectral dimension
is matched to the mass spectral peak width. Ions are detected
by the presence of local maxima in the filtered data matrix.
The 2D convolution filter is normalized so that an apex value
gives an approximate estimate of ion intensity. An ion is
considered to be detected if the filtered apex intensity is
above a threshold. The location of the apex in the filtered LC-
MS matrix determines the retention time and m/z ratio of
the ion. The intensity of the ion is obtained by extracting a
chromatographic profile, centered on the ion’s m/z ratio. The
ion’s intensity is the integrated area under the resulting 2D
chromatographic peak. The processed data are further
reduced with a de-isotoping and charge-state reduction algo-
rithm to provide a final inventory list of time-aligned mono-
isotopic mass measurements. Each precursor and product
ion is annotated with a monoisotopic mass, an intensity sum
from all isotopes and charge states, and an apex retention
time. Product ions are time-aligned to their parent precursor
ion if their apex retention time is within the time associated
to one-tenth of the precursor ions chromatographic peak
width at half-height (typically 61 scan).

3.2 DDA and LC-MSE acquisition of a simple and
complex protein mixture
Figure 1. Time-alignment correlation principle of precursor and
product ions. Chromatographic profile precursor and product ion A tryptic digest of a simple protein mixture of four proteins,
ions, (A) chromatographic profile for the precursor peptide see experimental section, was analyzed in triplicate by both
obtained during the low energy MS experiment, (B) chromato- data directed LC-MS/MS and LC-MSE. The DDA MS survey
graphic profile for all associated fragment ions generated during chromatograms and the low energy LC-MSE chromatograms
the elevated energy MS experiment (only one extracted isotopic
from the three replicate injections each are illustrated in
mass extracted of a single fragment ion is shown). (C) Chromat-
ographic peak characteristics: start (a); end (b); apex retention
Fig. S1 of Supporting Information, respectively. The profiles
time (c); width at half maximum for both precursor and asso- of the BPI chromatograms are similar between the two
ciated fragment ions (d plus e). The ratio of d over e is a measure modes of data acquisition, suggesting that the same peptides
for the chromatographic peak asymmetry. are sampled reproducibly within and across the two different
experiments. However, one distinguishing characteristic
seen in the low energy precursor ion survey spectra of the
aligned and correlated to the appropriate parent precursor two experiments is the consistently higher signal intensity in
ion. The culmination of this process results in an inventory the three replicate LC-MSE acquisitions. The data-directed
of all detected precursor ions with their associated product LC-MS/MS experiments were configured to select the three
ions. most intense ions for interrogation by MS/MS after each MS
The inventory lists are generated by processing the low survey scan. Therefore, the mass spectrometer was config-
and elevated energy LC-MS continuum data with a 3D peak ured to dedicate most of its time to the MS/MS mode of
detection algorithm. Ion detection is essentially accom- acquisition. The MS survey scan is thus sampled less fre-
plished by convolving a 3D LC-MS data matrix with a 2D fil- quently and as such the MS signal intensity of common
ter. An LC-MS data matrix is formed from a series of mass precursor ions is lower in the data directed method of acqui-
spectral scans, sampled uniformly in time. In such a matrix, sition.
an ion appears as a 3D Gaussian peak. In the absence of A similar comparison can be made between the BPI
detector noise, the location of each apex can be used to detect chromatograms generated from the triplicate analyzes of the
the ion and to determine retention time and m/z. The pres- same four proteins in the presence of a background of an E.
ence of noise gives however rise to multiple local maxima for coli lysate protein digest. The quantity of the four protein
each ion, so a simple apex-location scheme applied to the digest mixture was identical to the study above, and the
unfiltered data will produce spurious results. To reduce, and sample was analyzed in triplicate by both LC-MS/MS and LC-
to essentially, eliminate over counting, the 3D data matrix is MSE. The BPI chromatograms of both experiments are
convolved with a 2D convolution filter. The width of the filter shown in Fig. S2 of Supporting Information, and reflect
in the time dimension is matched to the chromatographic similar trends as observed with the simple protein mixture.



3.3 Comparison of results from DDA and LC-MSE The number of detected precursor ions reported in the
replicate LC-MSE analyses was 1290, 1249, and 1219, respec-
The results from the DDA and LC-MSE acquisition are pre- tively. Confirmation of a detected precursor ion matching to a
sented in the heat map shown in Fig. 2 and Figs. S3a–c of given peptide sequence within the LC-MSE experiments is
Supporting Information. The three columns on the left hand based solely on the presence/detection of a calculated mono-
side of these particular figures illustrate 153 DDA peptide isotopic mass within 610 ppm and 1 min in retention time to
identifications to the four standard proteins. These identifi- a DDA identification, with a minimum of three product ions
cations represent the combined results for all peptide identi- (620 ppm) to match. An intermethod 1 min time tolerance
fications from the three replicate DDA experiments. Note was used to account for the fact that a DDA retention time
that no single DDA experiment identified all of the peptides reflects the start of the MS/MS experiment and not the actual
listed. A green box and a value of one illustrates whether a chromatographic peak apex. Comparing the precursor ion
peptide sequence was identified for a particular DDA injec- masses and associated retention time measurements from
tion. A red box and a zero indicate that the peptide was not the three replicate LC-MSE experiments, using the same tol-
identified. For an LC-MSE experiment, a green box and a one erances as for the DDA data, also indicates a high degree of
represent the presence of a monoisotopic precursor ion mass consistency, approximately 90%, between replicate experi-
in the LC-MSE precursor/product ion table within 610 ppm ments. Interestingly, the intersection between the DDA and
and 61 min (two times the width of a chromatographic peak LC-MSE data sets also indicated a relative high degree of
at base), and with a minimum of three product ions within similarity, approximately 70%. More specifically a total of 506,
620 ppm, to that of an identified DDA peptide sequence. A 514, and 517 precursor ions from the LC-MSE experiments
red box and a zero indicate that there was no precursor ion in were found within the previously mentioned 733, 724, and
the LC-MSE precursor/product ion table within the chosen 740 MS/MS experiments from the DDA data based solely
match criteria. The number of MS/MS acquisition events for upon m/z and time. The latter was achieved by using the same
the three replicate DDA experiments were 733, 724, and 740, matching criteria as described in the previous paragraph. A
respectively. Comparing the m/z values and retention time detailed overview of the LC-MSE detections and identifications
measurements from the three replicate DDA analyzes is provided in the Table S2 of Supporting Information. Each
revealed a high degree of consistency between the acquisi- peptide detection/identification is annotated with the asso-
tions. Approximately 89% of the selected masses were at the ciated retention time (peak apex), intensity, charge state, and
same m/z value (610 ppm) and at the same retention time replication rate. The results shown in Tables S1 and S2 of
(630 s). A detailed overview of the DDA detections and Supporting Information, and the heat map representations
identifications is provided in Table S1 of Supporting Infor- shown in Fig. 2 and Figs. S3a–c of Supporting Information,
mation. Each peptide detection/identification is annotated illustrate the commonality of the data between the acquisition
with associated retention time (start time MS/MS acquisi- methods. On average, 151 of the 153 possible monoisotopic
tion), intensity, and replication rate. peptide masses (approximately 99%) were detected in every

Figure 2. Heat-map representation of the identified and detected peptides to alcohol dehydrogenase from three replicate LC-MS/MS and
three replicate LC-MSE and experiments in the presence and absence of 0.5 mg E. coli tryptic digest. Experiments that generated appropriate
precursor and product ion information for the corresponding peptide of the proteins of interest are indicated by a green box and a 1 and
those that did not contain the data are indicated by a red rectangle and a 0.


Proteomics 2009, 9, 1683–1695 1689

single replicate LC-MSE experiment. Note that the detection respect to the presence of detectable precursor and product
of the correlated DDA and LC-MSE accurate mass precursor ion masses, the number of detectable product ions and the
and product ions at the appropriate retention time does not observed fragmentation patterns. The subsequent identifica-
constitute identification of the LC-MSE data at this stage. It tion of the detected peptide precursor ions and their asso-
should, however, also be noted that not all detected LC-MSE ciated time-aligned fragments will be described in detail in a
features are shown in Fig. 2 and Figs. S3a–c of Supporting subsequent manuscript that describes the databank search
Information, but only those in common with the combined algorithm [29]. This manuscript illustrates that the same
DDA results. ions are detected by both acquisition methods and that mul-
The results from the DDA and LC-MSE experiments tiplexed data can be aligned by retention time. In addition,
from the simple four protein mixture in the presence of a the S/N of LC-MSE data at both the precursor and product ion
tryptically digested cytosolic E. coli background are illustrated level is generally greater than that of the matching DDA data.
in the right-hand side of Fig. 2 and Figs. S3a–c of Supporting The number of MS/MS experiments for the three repli-
Information. The results show a significant decrease in the cate DDA experiments of the four protein mixture spiked
number of identifications to the 153 possible detectable pep- into the enzymatically digested cytosolic E. coli background
tides of the four protein mixture utilizing the DDA acquisi- was 1586, 1589, and 1651, respectively. Interestingly, the
tion method. Previously, 137 of the 153 peptides of the four replication on m/z and retention time between these three
protein mixture were identified in each individual DDA experiments was again relatively high, approximately 83%,
experiment. However, after the addition of the E. coli lysate, see Table S3 of Supporting Information. These results chal-
this number decreased drastically from 137 to 70. This lenge the common perception that a DDA method selects
represents slightly less than a 50% decrease in the number of precursor ions for MS/MS in a serendipitous fashion. It is
peptides belonging to the four protein digest that were sam- often suggested and claimed that these finding are due to the
pled. With the LC-MSE acquisition method, an average of 141 instrumentation and/or the acquisition parameters used for
of the 153 peptide monoisotopic masses of interest was DDA experiments. Therefore, a comparative study has been
detected, applying the aforementioned match criteria. A conducted that includes replicate DDA injections on a num-
summary of the results is provided in Table 1. This suggests ber of different tandem mass spectrometer platforms with
that even in the presence of a very complex background, the various sampling rate, scan speed and DDA characteristics.
data independent acquisition method still detected the same In all instances, the same LC system and on-column load of
peptide precursor ions, at the same retention time. The cytosolic E. coli digest were used. It was found that the repli-
presence of accurately mass measured precursor and prod- cation rates, without the use of include or exclude lists, stea-
uct ions at the appropriate retention times implies that there died after two replicate experiments at approximately 67%
is a high degree of similarity between the two data sets with and that they were instrument platform independent. The

Table 1. Fractional and replication fractional number of detected peptides by data dependent LC-MS/MS DDA and data independent LC-
MSE for a four protein mixture in the absence and presence of a complex biological E. coli digest background

Protein

Alcohol Enolase Glycogen Serum albumin
dehydrogenase phosphorylase

Maximum achievable 20 21 67 43
peptide detectionsa) E E E
DDA LC-MS DDA LC-MS DDA LC-MS DDA LC-MSE

Fractional detection four protein mixture
Inj. 1 0.90 1.00 0.90 1.00 0.88 1.00 0.93 0.99
Inj. 2 0.90 1.00 0.81 1.00 0.84 1.00 0.87 0.99
Inj. 3 0.90 1.00 0.81 1.00 0.84 1.00 0.82 0.97
Replicating detectionsb) 0.95 1.00 0.86 1.00 0.91 1.00 0.82 0.99
Fractional detection four protein mixture in E. coli
Inj. 1 0.50 0.95 0.43 0.81 0.44 0.88 0.42 0.87
Inj. 2 0.50 0.90 0.48 1.00 0.26 0.93 0.37 0.94
Inj. 3 0.65 1.00 0.57 1.00 0.23 1.00 0.57 0.96
Replicating detectionsb) 0.50 0.95 0.52 1.00 0.23 0.95 0.40 0.96

a) Maximum number of peptides that can be identified based on the combined results of the three replicate DDA injections.
b) Fractional number based on replicating identifications (!2 out 3 replicate experiments).



latter will be the subject of a separate manuscript. The num- proportional to the log recorded precursor ions intensity. The
ber of reported precursor ions for the three replicate LC-MSE results shown in Fig. 3 show an increase in precursor ion
experiments for the four protein mixture spiked into the intensity of the LC-MSE ion detections over their DDA coun-
enzymatically digested cytosolic E. coli sample was 26 902, ter parts. Ramos and coworkers [23] have recently reiterated
27 015, and 25 943, respectively, see Table S4 of Supporting these observations. They report that parallel fragmentation
Information. These numbers are significantly higher than experiments produce product ion spectra with substantially
the number of MS/MS experiments for the DDA LC-MS/MS increased signal intensities, attributed to the sampling of
analyses since there is no precursor ion selection applied. As virtually all the ions generated by ESI. The results show that
a result, all detectable precursor and fragment ions are effi- as a consequence of the increased signal intensity of the LC-
ciently and continuously sampled. The replication on MSE generated product ion spectra, the total number of
(M 1 H)1 and retention time between these three experi- detectable fragment ions is increased and a more compre-
ments was approximately 70%. A number very similar to the hensive characterization at both the peptide and parent pro-
results obtained for the DDA LC-MS/MS data. tein level provided.
The increase in sensitivity for the multiplexed acquisi-
tion method is illustrated in Fig. 3 by superimposing the 3.4 Time-alignment product/fragment ions
distributions of the corresponding DDA and LC-MSE peptide
pairs, in the presence of the digested cytosolic E. coli pro- Precursor accurate mass and retention time are not always
teins. The solid red dots represent the DDA identifications sufficiently unique to provide unambiguous peptide identi-
and the solid blue dots the LC-MSE detections. The grey cir- fication. Figures 4a–h depicts deconvoluted product ion
cles are E. coli peptide identifications. The size of the spot is spectra and MS survey chromatograms generated by both

Figure 3. Scanning technique, i.e., DDA (red) and LC-MSE (blue), common identified peptides pairs in the presence of 0.5 mg E. coli tryptic
digest. The spot size is proportional to the logarithm of the precursor ion intensity; grey circles illustrate background E. coli peptide iden-
tifications.


Proteomics 2009, 9, 1683–1695 1691

Figure 4. Deconvoluted product ion spectrum of tryptic albumin peptide LVNELTEFAK spectra generated by DDA (a) and the corresponding
time-aligned LC-MSE (b) product ion spectra in the four protein mixture. Spectra (d) and (e) show similar information for tryptic albumin
peptide RPCFSALTPETYVPK. Spectra (f) and (g) are as (a) and (b); however, now in the presence of 0.5 mg E. coli tryptic digest. Panes (c) and
(h) show the low energy LC-MSE chromatograms and coeluting behavior of both albumin peptides in the absence (c) and presence (h) of an
E. coli digest background. Fragment ion color legend: red = y ion; blue = b ion; green = immonium ion or neutral loss of NH3 or H2O;
gray = not identified; magenta = precursor or fragment ion assigned to a coeluting peptide.

acquisition methods. Figure 4a shows the identified DDA 1163.6305 and (M 1 H)1 1880.9124 precursor ions detected
product ions from one of the replicate injections from the by the LC-MSE acquisition method is a mere 0.04 min.
four protein mixture to tryptic peptide LVNELTEFAK from Comparing the 164 product ions from the DDA spectrum of
albumin. Figure 4b illustrates the corresponding product peptide sequence RPCFSALTPETYVPK from albumin with
ions in the time-aligned spectrum from one of the LC-MSE the 189 product from the LC-MSE time-aligned ions from
experiments. A very similar number of detected ions is precursor ions (M 1 H)1 1163.6305 and (M 1 H)1
obtained from both acquisition methods by extracting the 1880.9124 resulted in the detection of an additional 37 cor-
product ions from both spectra, see Table S5 of Supporting responding product ions within a 620 ppm tolerance. This
Information. The DDA and LC-MSE spectra comprised 146 confirms that the LC-MSE data processing algorithms were
and 189 product ions, respectively. A comparative analysis of capable of the correct detection and time-alignment of the
the product ion lists of the two spectra with a mass precision appropriate product ions.
of 620 ppm resulted in an intersection of 26 product ions. Figure 4f shows the DDA spectrum associated to the
A more careful perusal of the LC-MSE spectrum in Fig. 4b same LVNELTEFAK tryptic peptide from albumin present in
illustrates the presence of some relatively high intensity the E. coli background, whereby Fig. 4g shows the detected
nonidentified product ions. Figure 4c shows a section of the and time-aligned LC-MSE product ions to the precursor ion
low energy LC-MSE chromatogram of the four protein mix- of the calculated monoisotopic mass of 1163.6309 at the ap-
ture and indicates the presence of a second precursor ion of propriate retention time. Figure 4h illustrates a section of the
(M 1 H)1 1880.9124 within one-tenth of the time associated low energy LC-MSE chromatogram of the four protein mix-
to the chromatographic peak width at half-height of ture in the presence of the digested cytosolic E. coli proteins.
(M 1 H)1 1163.6299. As such, the LC-MSE spectrum, since As to be expected, the second albumin peptide RPCFSALT-
no precursor isolation was applied, will share some product PETYVPK of precursor ion mass (M 1 H)1 1880.9174 is
ions from both precursor ions. Inspection of the DDA search present within one-tenth of the time associated to the chro-
result shown in Fig. 4d reveals that the coeluting precursor matographic peak width at half-height of the companion
ion could be identified to the tryptic peptide sequence precursor ion of (M 1 H)1 1163.6309. As previously stated,
RPCFSALTPETYVPK from albumin. Figure 4e depicts the they will therefore also share certain product ions. Note that
corresponding time-aligned and detected product ions from the retention times, Fig. 4c versus Fig. 4h, were most likely to
the LC-MSE data to precursor ion (M 1 H)1 1880.9124. Note be affected due to the presence of a very large number of E.
that the time difference of the apices of the (M 1 H)1 coli peptides. Also note the presence of over 14 other E. coli



peptides of varying intensity that coelute with the two albu- ments and retention times, provide an additional dimension
min peptides within a time window of approximately 6 s. A of specificity for a given tryptic peptide map of a protein. The
simple perusal of Fig. 4a–h illustrates a very high degree of relative intensity measurements of the tryptic peptides iden-
similarity in both the fragmentation pattern and the number tified to glycogen phosphorylase are shown in Fig. 5 from
of product ions detected by the two acquisition methods. Al- both the LC-MSE and the LC-MS/MS acquisitions. The re-
though the magnitude of the increase varies, it is clear that producibility of these integrated peak measurements are
the product ion intensities in the LC-MSE data sets are higher depicted by the error bars, which correspond to the calcu-
than their DDA counterparts. This is due to the acquisition lated intensity RSD errors. The peptides have been ordered
of LC-MSE data on all isotopes and charge-states across the by decreasing intensity as determined from the LC-MSE data
precursor ions chromatographic peak width and is illustrated acquisition method. From this plot, a smooth trend of high
in more detail in the next paragraph. (, = 1.0 and . = 0.666), medium (,0.666 and .0.333), and
low (, = 0.333 and .0) ionizing tryptic peptides can be
3.5 Relative intensity profiles of tryptic peptides from observed. Similar observations can be seen for the other
LC-MSE data three proteins and are shown in Figs. S4a–c of Supporting
Information. This behavior is consistent with previously
The ability to acquire both the precursor ion and associated published data [26, 30] and can be used to facilitate the
fragment ion data throughout the entire peak width of all interrogation of complex protein samples for any specific
detected peptides in a consistent and reproducible fashion protein of interest, once its tryptic peptide ion map has been
enables the use of integrated peak areas as an additional characterized. For example, in cases where one is interested
physiochemical attribute that can be used for the characteri- in identifying low levels of the same protein one would initi-
zation of all identified proteins [29]. The relative relationship ally look for the top two or three best ionizing peptides to the
of the intensity measurements of all identified tryptic pep- protein along with their corresponding product ion spectra
tides, along with the associated accurate mass measure- and also confirm the absence of the lowest ionizing peptides.

Figure 5. Intensity profiles of the tryptic peptides identified to glycogen phosphorylase. The absolute LC-MSE (dark grey) and relative LC-
MS/MS DDA (light grey) profiles, including intensity measurement errors, are shown for the 46 characterized peptides of interest.


Proteomics 2009, 9, 1683–1695 1693

These types of profiles can be generated for a panel of pro- 4 Conclusions
teins and used for the investigation of a particular complex
protein sample by limiting the survey to a specific set of An LC-MS data acquisition method comparison is described
proteins involved in a biologically, or phenotypically, relevant to illustrate that the information content of data independent
pathway [26]. The integrated DDA peak intensities for the acquisition LC-MS experiments is comparable to that of
same peptides are also plotted with their corresponding DDA LC-MS/MS acquisitions. The utilized data-independent
measurement error. Due to the inherent more random na- LC-MSE method in this paper could be considered to be a
ture of the LC-MS/MS acquisition method, there is no corre- parallel acquisition approach, resulting in a vastly improved
lation with the observed peak intensities. This suggests that mass spectrometer duty cycle. Unlike traditional data
precursor intensity information from the tryptic peptides dependant MS/MS approaches, the method does not require
obtained from DDA LC-MS/MS experiments cannot be uti- real time decisions to be made on which precursor ions to
lized as quantitative features for the characterization of a select, such as MS/MS switching thresholds or the recogni-
given protein. tion and subsequent selection of specific charge states for
As previously mentioned, product ion spectra for every fragmentation. As a consequence, partial sampling of chro-
detected precursor are obtained at their chromatographic matographic peaks does not occur, eliminating some of the
apex for optimum sensitivity, which is a direct con- drawbacks associated with current data directed LC-MS/MS
sequence of the successful monitoring of every peptide approaches. The LC-MSE method acquires precursor and
across its chromatographic elution. Compared to conven- product ion data on all charge-states of an eluting peptide
tional tandem DDA LC-MS/MS, where individual peptides across its entire chromatographic peak width, providing
are selected and fragmented serially, LC-MSE allows data more comprehensive precursor and product ion spectra.
from multiple peptide species to be collected simulta- Moreover, with a data independent acquisition, the combi-
neously, capturing all of the precursor and fragment ion nation of a high-peak capacity chromatographic separation
information without bias, and potentially with higher with high sampling-rate orthogonal acceleration TOF mass
throughput. Masselon et al. [18, 31] have demonstrated spectrometer provides a rapid and parallel approach for gen-
how accurate mass instruments can be used to increase erating peptide precursor and product ion detections on all
the throughput of peptide identifications using a multi- eluting species across the chromatographic peak profiles.
plexed approach as the basis for faster and more sensitive This would not have been afforded by a more traditional data
peptide identifications in LC-MS-based experiments. An dependent approach because of the inherent undersampling
LC-MSE experiment integrates the ability to measure both of the method. A data independent approach is therefore
accurate mass and retention time, whilst generating prod- believed to be more suited for relative and absolute quantifi-
uct ion spectra having consistently higher intensity than cation, in both label-free and stable isotope labeled quantita-
that of the corresponding LC-MS/MS product ion spectra. tive proteomics experiments. Furthermore, a data independ-
Also noted by Masselon et al. [18, 31], it is expected that ent acquisition method is likely to be more reproducible
new statistical approaches, and more sophisticated scoring across instrument platforms when analyzing similar sam-
systems, will evolve to properly manage this type of multi- ples. The unbiased and reproducible nature of these experi-
plexed data. Using the inherent intensity profile informa- ments will, in the authors’ opinion, promote the increased
tion of the detected precursors and their associated product use of data-independent, parallel methods, for the analysis of
ions obtained from this method, it is expected that low complex biological samples.
abundance proteins can be more effectively identified in Low energy, parent precursor, and elevated-energy,
complex mixtures. product ions share the same chromatographic profile and
In a typical database search, however, the mass accu- apex retention-time, which provides the capacity to correlate
racy of the detected precursor and fragments as well as the them and provide additional specificity to the experiment.
number of matching fragments are some of the criteria However, precursor ions will and do experience coelution to
used to determine a positive peptide identification. As the a degree. This degree of coelution is acknowledged and
complexity of the data increases, more stringent criteria are addressed during the processing, which is in contrast to
needed to improve the accuracy and specificity of the data directed/dependant LC-MS/MS experiments, where the
search results [32]. The reproducibility of the mass and coincident fragmentation of precursor ions in the collision
intensity measurements of the peptides, and their asso- cell is typically not addressed. Despite this, certain coeluting
ciated product ions, in conjunction with an additional di- product ions, the elevated-energy ions that cannot be exclu-
mension of information (time) provides a higher degree of sively assigned to a precursor ion, will initially be shared
specificity and selectivity for conducting proteomic studies. between multiple precursors. However, with the afforded
An hierarchical database search strategy has been devel- mass measurement accuracy on both precursor and prod-
oped that incorporates these and other attributes to effi- ucts ions and the subsequent database search strategy
ciently process LC-MSE data, providing both high sensitiv- employed, these additional product ions present within the
ity and specificity, and is the subject of a further manu- spectrum have a very minor effect on the identification of
script [29]. the peptides.



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