An Integrated Approach to Improve
Sequence Coverage and Protein
Identification by Combining LC-MALDI
MS/MS and Nano-LC/MS/MS
Application Note
Ning Tang and Christine Miller
Agilent Technologies

This material was presented in a poster at the 53rd ASMS Conference on Mass
Spectrometry in 2005. Research presented in posters at scientific conferences
may include results from instruments or products that are not yet commerciallyavailable.

Introduction

Materials

Electrospray ionization (ESI) and matrix-assisted laser
desorption ionization (MALDI) have proven to be complementary as different peptides are typically seen withthe two ionization techniques, however, very little was
known about the characteristics of the peptides thatprefers each ionization technique. Coupling of theRP-HPLC with MALDI via a MALDI spotting device has
provided an automated and robust solution for analyzing
medium complexity samples using MALDI. In this study,
we have investigated the improvements in proteome
coverage for complex protein samples by combining
nano-LC/MS/MS with LC-MALDI/MS/MS. The integrated
approach with better sequence coverage facilitated protein identification.

HPLC grade solvents were obtained from Burdick &Jackson. Human serum was purchased from Genomics
Collaborative (Cambridge, MA). It was from a healthy
anonymous male donor. The immunodepletion column
and reagents, the α-cyano-4-hydroxy-cinnamic acid(CHCA) matrix solution, and the in-gel digestion kit
were from Agilent Technologies.

Experimental

Offline SCX separation

Three dimensions of separations were employed to simplify the human serum sample before it was analyzed by
the mass spectrometry (see Figure 1). The first dimension of separation was immunodepletion of six of themost abundant proteins in human serum using an antibody column. The second dimension was strong cationexchange (SCX) fractionation of the tryptic peptides.
The third dimension was reversed-phase chromatography of the SCX fractions before mass spectrometry
analysis.

Offline capillary LC prefractionation was done using aZorbax Bio-SCX Series II column, 50 x 0.8 mm, with anammonium formate gradient. The gradient went from
0–100 mM in 120 minutes, then at 500 mM at 134 minutes and finally to 1 M at 140 minutes. Fifty fractions
were collected. Fractions were dried, washed twice with
H2O to remove the ammonium bicarbonate, and thendried before LC-MALDI and direct MALDI analysis.

Immunodepletion
Human serum was immunodepleted using the Agilent
Multiple Affinity Removal system (MARS), whichremoves six of the highest abundance proteins in serum.
The depleted serum was then denatured, reduced, alkylated and digested with trypsin.

An Integrated Approach to Improve Sequence Coverage and Protein Identification
by Combining LC-MALDI MS/MS and Nano-LC/MS/MS

Agilent Technologies

LC-MALDI MS/MS
Human serum sample

2500
2000
1500
1000
5000MARS02468101214Denature, reduce
Alkylate, digest

Samples were separated using a capillary HPLC
equipped with a thermostatted micro fraction collector/spotter (Agilent Technologies). The flow rate was 1 µL/min.
The matrix CHCA (2 mg/mL diluted with 50% 2-propanol/
1% acetic acid) was added to the sample at 1 µL/min
using a syringe pump connected to a T-connector. The
spotter spotted every minute with 2 µL of total volume
on the MALDI target. The target was then analyzed using
the LC/MSD Trap XCT Plus ion trap mass spectrometerwith a PDF-MALDI source.

Nano-LC/MS/MS
All LC/MS analyses were done using the Agilent Protein
Identification Solution which consisted of an Agilent
1100 Series nanoflow liquid chromatograph (LC) system
coupled to an Agilent 1100 Series LC/MSD Trap XCTPlus.

Database search

102030min

SCX fractionation

Data processing was done using Spectrum Mill MS proteomics workbench software. The spectra were searchedagainst the IPI-human database using carbamidomethylation as the Cys modification. The search was donewith three iterative cycles. The first search was done inthe identity mode against the IPI-human database withtrypsin cleavage specified and 2 missed cleavages
allowed. The validated search results were saved as a
new database for the next search cycle. The second
search cycle was done against the smaller saved results
database to search for single KQMSTY modifications.
The results were then validated and saved. The third
search cycle was done against the saved results databasewith the enzyme selection set to none.

Results
Comparison of LC-MALDI MS/MS with direct
MALDI MS/MSDryWash

Direct MALDI

LC-MALDI

Figure 1. Experimental design

2

Nano-LC/MS/MSThe SCX fractions were run by both MALDI MS/MS and
LC-MALDI MS/MS. The number of proteins and peptides identified by each technique were summarized inTable 1. LC-MALDI identified 3–4 fold more proteins and
5–6 fold more peptides compared to direct MALDI.
Sequence coverage of identified proteins increased by
LC-MALDI, which also provided better confidence in theprotein ID.

An Integrated Approach to Improve Sequence Coverage and Protein Identification
by Combining LC-MALDI MS/MS and Nano-LC/MS/MSAgilent Technologies

Table 1. Comparison of the number of proteins and peptides
identified by LC-MALDI MS/MS and direct MALDI MS/MS

Table 2. Comparison of the number of proteins and peptides
identified by LC-MALDI MS/MS and nano-LC/MS/MS# of proteins# of peptides# of proteins# of peptides

Direct MALDI

2356

Nano-LC/MS/MS

89

876

LC-MALDI

76313

LC-MALDI

76313

Combination

1291044

Combine results from LC-MALDI MS/MS andnano-LC/MS/MS
We have identified 129 proteins in total combining theresults from both techniques. Out of the 129 proteins, 40of them were identified by both techniques. Forty proteins were only identified by LC-MALDI MS/MS and 49
proteins were uniquely identified by nano-LC/MS/MS.
Combining the two techniques has increased the numberof proteins identified as well as sequence coverage anddatabase search scores.

49

40

Each technique identified unique peptides among theproteins found by both methods. Combining the results
increased the sequence coverage of the proteins. Figure
3 is an example of Spectrum Mill database search resultsof one of the protein that was identified as inter-alphatrypsin inhibitor heavy chain H2 precursor.

40
Figure 3. Spectrum Mill display of all the peptides identified by
LC-MALDI MS/MS and nano-LC/MS/MS

Discussion

Nano-LC/MS/MS

LC-MALDI MS/MS

Figure 2. Combining the results from nano-LC/MS/MS andLC-MALDI MS/MS

Comparison of LC-MALDI MS/MS with nano-LC/MS/MS
The number of proteins and peptides identified by eachtechnique were summarized in Table 2. LC-MALDI
MS/MS and nano-LC/MS/MS identified similar numberof proteins. However, nano-LC/MS/MS identified 2–3 foldmore peptides, therefore, it provided higher sequence
coverage and more confident protein ID. Combining thetwo techniques, we have identified more proteins withmore peptides.

We compared our results with the nonredundant list
developed by Anderson et al. from a combination of four
separate sources (see Table 3). Thirty seven of the proteins we identified were also identified by those other4 sources. These proteins we have identified with relatively high sequence coverage and Spectrum Mill database search scores. They represented the high abundant
proteins and are more likely to be identified most of thetime regardless of the different separation techniquesand mass spectrometry that was used. Twenty one proteins we identified were also identified by three othersources. They have lower sequence coverage and Spectrum Mill scores. Twelve proteins were identified by twoother sources and fifty nine proteins were identified byless than two other sources. They have relatively low
sequence coverage and low spectrum mill scores.

3

An Integrated Approach to Improve Sequence Coverage and Protein Identification
by Combining LC-MALDI MS/MS and Nano-LC/MS/MS

Agilent Technologies

Table 3. Correlation of Agilent results with nonredundant list comprised of data from four separate sources

ID by othersources# ofproteins

Average #of peptides
per protein

Averagesequence
coverage (%)

Average
Spectrum
Mill scores

437

1526210321

1112143

2123

1136

<259

4

740

Conclusions

References• LC separation coupled with MALDI has significantly
increased the number of proteins identified.

1. Nagele, E. and Vollmer, Coupling of nanoflow liquid
chromatography to matrix-assisted laser desorption/ionization mass spectrometry: real-time liquid
chromatography run mapping on a MALDI plate,
M. Rapid Commun. Mass spectrum. 2004; 18: 3008

• LC-MALDI MS/MS and nano-LC/MS/MS have eachidentified unique peptides among the proteins foundby both techniques and combining the results gives
increased sequence coverage for most of the proteins.
• Each technique has identified unique proteins. LCMALDI MS/MS has identified 40 unique proteins andnano-LC/MS/MS has identified 49 unique proteins.
Combining both techniques, 129 proteins were identified.

2. Anderson, N. L. et al., The human plasma proteome,Mol. Cell. Proteomics, 2004; 3.4: 311–326.

• Combining both techniques increased the confidence
in the protein identification and gave the optimum
sequence coverage for the proteins.

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