Proteomics 2009 V9p1683

693 views

Published on

Database search engine for data independent proteomics analysis

0 Comments
0 Likes
Statistics
Notes
  • Be the first to comment

  • Be the first to like this

No Downloads
Views
Total views
693
On SlideShare
0
From Embeds
0
Number of Embeds
24
Actions
Shares
0
Downloads
5
Comments
0
Likes
0
Embeds 0
No embeds

No notes for slide

Proteomics 2009 V9p1683

  1. 1. 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
  2. 2. 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 © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
  3. 3. 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 © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
  4. 4. 1686 S. J. Geromanos et al. Proteomics 2009, 9, 1683–1695 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- © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
  5. 5. 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. © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
  6. 6. 1688 S. J. Geromanos et al. Proteomics 2009, 9, 1683–1695 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. © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
  7. 7. 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). © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
  8. 8. 1690 S. J. Geromanos et al. Proteomics 2009, 9, 1683–1695 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. © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
  9. 9. 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 © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
  10. 10. 1692 S. J. Geromanos et al. Proteomics 2009, 9, 1683–1695 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. © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
  11. 11. 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. © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
  12. 12. 1694 S. J. Geromanos et al. Proteomics 2009, 9, 1683–1695 Mass spectrometric identification information is cur- [8] Hoopmann, M. R., Finney, G. L., MacCoss, M. J., High-speed rently populated into proteomic repositories such as Pepti- data reduction, feature detection, and MS/MS spectrum quality assessment of shotgun proteomics data sets using deAtlas, The Global Proteome Machine and PRIDE, sup- high-resolution mass spectrometry. Anal. Chem. 2007, 79, porting the concept of using peptide database libraries as a 5620–5632. means to identify and/or validate the presence of peptides [9] Luethy, R., Kessner, D. E., Katz, J. E., MacLean, B. et al., Pre- and proteins from the compilation of empirically derived cursor-ion mass re-estimation improves peptide identifica- data. The information ultimately gained from LC-MSE tion on hybrid instruments. J. Proteome Res. 2008, 7, 4031– experiments, i.e., the identification of peptides and proteins, 4039 including all the precursor ions and product ions matched to [10] Carr, S., Aebersold, R., Baldwin, M., Burlingame, A. et al., every protein, each with its monoisotopic mass, retention- The need for guidelines in publication of peptide and protein time, charge-states, and summed intensities, allows for the identification data: Working Group on publication guide- continuous accumulation of both the qualitative and quanti- lines for peptide and protein identification data, Mol. Cell. tative information in these types of repositories, with the Proteomics 2004, 3, 531–533. benefit of applying specific (bio) analytical reproducibility [11] Wilkins, M. R., Appel, R. D., Van Eyk, J. E., Chung, M. C. M. et measures. The qualitative and quantitative results obtained al., Guidelines for the next 10 years of proteomics, Prote- from these studies will serve to further advance the under- omics 2006, 6, 4–8. standing of proteomics and will be used to address important [12] Chamrad, D., Meyer, H. E., Valid data from large-scale pro- biological questions. teomics studies, Nat. Methods 2005, 2, 647–648. [13] Anderson, N. L., Polanski, M., Pieper, R., Gatlin, T. et al., The Human plasma proteome – A nonredundant list developed The authors would like to acknowledge the valuable con- by combination of four separate sources, Mol. Cell Prote- omics 2004, 3, 311–326. tributions of Timothy Riley throughout the development of this work. We also thank Martha Stapels, Michael Nold and Joanne [14] Miyamoto, M., Yoshida, Y., Taguchi, I., Nagasaka, Y. et al., In- depth proteomic profiling of the normal human kidney glo- Connolly for their ideas and contributions throughout the editing merulus using two-dimensional protein prefractionation in of this manuscript. combination with liquid chromatography-tandem mass spectrometry. J. Proteome Res. 2007, 6, 3680–3690. The authors have declared no conflict of interest. [15] Bateman, R. H., Carruthers, R., Hoyes, J. B., Jones, C. et al., A novel precursor ion discovery method on a hybrid quadru- pole orthogonal acceleration time-of-Flight (Q-TOF) mass 5 References spectrometer for studying protein phosphorylation. J. Am. Soc. Mass Spectrom. 2002, 13, 792–803. [1] Strupat, K., Karas, M., Hillenkamp, F., Eckerskorn, C., Lott- [16] Silva, J. C., Denny, R., Dorschel, C. A., Gorenstein, M. V. et speich, F., Matrix-assisted laser desorption ionization mass al., Quantitative proteomic analysis by accurate mass reten- spectrometry of proteins electroblotted after polyacrylamide tion time pairs. Anal. Chem. 2005, 77, 2187–2200. gel electrophoresis. Anal. Chem. 1994, 66, 464–470. [17] Williams, E. R., Loh, S. Y., McLafferty, F. W., Cody, R. B., [2] Mann, M., Hojrup, P., Roepstorff, P., Use of mass spectromet- Hadamard transform measurement of tandem fourier- ric molecular weight information to identify proteins in transform mass spectra. Anal. Chem. 1990, 62, 698–703. sequence databases. Biol. Mass Spectrom. 1993, 22, 338–345. [18] Masselon, C., Anderson, G. A., Harkewicz, R., Bruce, J. E. et [3] McCormack, A. L., Schieltz, D., Eng, J. K., Goode, B. et al., al., Accurate mass multiplexed tandem mass spectrometry Direct analysis and identification of proteins in mixtures by for high-throughput polypeptide identification from mix- LC/MS/MS and database searching at the low femtomole tures. Anal. Chem. 2000, 72, 1918–1924. level. Anal. Chem. 1997, 69, 767–776. [19] Wilson, J., Vachet, R. W., Multiplexed MS/MS in a Quadru- [4] Washburn, M. P., Wolters, D., Yates, III, J. R., Large scale pole Ion Trap Mass Spectrometer. Anal. Chem. 2004, 76, analysis of the yeast proteome via multidimensional protein 7346–7353. identification technology, Nat. Biotech. 2001, 19, 242–247. [20] Venable, J. D., Dong, M.-Q., Wohlschlegel, J., Dillin, A., [5] Peng, J., Elias, J. E., Thoreen, C. C., Licklider, L. J., Gygi, S. P., Yates, III, J. R., Automated approach for quantitative analy- Evaluation of multidimensional chromatography coupled sis of complex peptide mixtures from tandem mass spectra. with tandem mass spectrometry (LC/LC-MS/MS) for large- Nat. Methods 2004, 1, 39–45. scale protein analysis: The yeast proteome. J. Proteome Res. 2003, 2, 43–50. [21] Purvine, S., Eppel, J.-T., Yi, E. C., Goodlett, D. R., Shotgun collision-induced dissociation of peptides using a time of [6] Ishihama, Y., Oda, Y., Tabata, T., Sato, T. et al., Exponentially flight mass analyzer. Proteomics 2003, 3, 847–850. modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of [22] Hoaglund-Hyzer, C. S., Li, J., Clemmer, D. E., Mobility label- sequenced peptides per protein. Mol. Cell. Proteomics 2005, ing for parallel CID of ion mixtures. Anal. Chem. 2000, 72, 4, 1265–1272. 2737–2740. [7] Geromanos, S., Freckleton, G., Tempst, P., Tuning of an electro- [23] Ramos, A. A., Yang, H., Rosen, L. E., Yao, X., Tandem parallel spray ionization source for maximum peptide-ion transmission fragmentation of peptides for mass spectrometry. Anal. into a mass spectrometer. Anal. Chem. 2000, 72, 777–790. Chem. 2006, 78, 6391–6397. © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
  13. 13. Proteomics 2009, 9, 1683–1695 1695 [24] Silva, J. C., Denny, R., Dorschel, C. A., Gorenstein, M. V. et spray tandem mass spectrometry data. Comp. Funct. al., Simultaneous qualitative and quantitative analysis of the Genom. 2004, 5, 61–68. E. coli proteome: A sweet tale. Mol. Cell. Proteomics 2006, 5, [29] Li, G.-Z., Vissers, J. P. C., Silva, J. C., Golick, D. et al., Data- 589–607. base searching and accounting of multiplexed precursor [25] Hughes, M., Silva, J. C., Geromanos, S. J., Townsend, C. A., and product ion spectra from the data independent analysis Quantitative proteomic analysis of drug-induced changes in of simple and complex peptide mixtures. Proteomics, 2009, mycobacteria. J. Proteome Res. 2006, 5, 54–63. 9, 1696–1719. [26] Vissers, J. P. C., Langridge, J. I., Aerts, J. M. F. G., Analysis [30] Silva, J. C., Gorenstein, M. V., Li, G.-Z., Vissers, J. P. C., Ger- and quantification diagnostic serum markers and protein omanos, S. J., Absolute quantification of proteins by signatures for gaucher disease. Mol. Cell. Proteomics 2007, LCMSE: A virtue of parallel MS acquisition. Mol. Cell. Prote- 5, 755–766. omics 2006, 5, 144–156. [27] Kraemer-Albers, E.-M., Bretz, N., Tenzer, S., Winterstein, C. [31] Masselon, C., Pasa-Tolic, L., Lee, S.-W., Li, L. et al., Identifi- et al., Oligodendrocytes secrete exosomes containing major cation of tryptic peptides from large databases using multi- myelin and stress-protective proteins: Trophic support for plexed tandem mass spectrometry: Simulations and axons? Proteomics Clin. Appl. 2007, 1, 1446–1461. experimental results. Proteomics 2003, 3, 1279–1286. [28] Skilling, J., Denny, R., Richardson, K., Young, P. et al., Prob- [32] Zubarev, R., Mann, M., On the proper use of mass accuracy Seq–A fragmentation model for interpretation of electro- in proteomics. Mol. Cell. Proteomics 2006, 6, 377–381. © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

×