Global Relative Quantification with Liquid Chromatography–Matrix-assisted Laser Desorption Ionization Time-of-flight (LC-MALDI-TOF)—Cross–validation with LTQ-Orbitrap Proves Reliability and Reveals Complementary Ionization Preferences

Abstract

Quantitative LC-MALDI is an underrepresented method, especially in large-scale experiments. The additional fractionation step that is needed for most MALDI-TOF-TOF instruments, the comparatively long analysis time, and the very limited number of established software tools for the data analysis render LC-MALDI a niche application for large quantitative analyses beside the widespread LCelectrospray ionization workflows.Here, we used LC-MALDI in a relative quantification analysis of Staphylococcus aureus for the first time on a proteome-wide scale. Samples were analyzed in parallel with an LTQ-Orbitrap, which allowed cross-validation with a well-established workflow. With nearly 850 proteins identified in the cytosolic fraction and quantitative data for more than 550 proteins obtained with the MASCOT Distiller software, we were able to prove that LC-MALDI is able to process highly complex samples. The good correlation of quantities determined via this method and the LTQ-Orbitrap workflow confirmed the high reliability of our LC-MALDI approach for global quantification analysis.Because the existing literature reports differences for MALDI and electrospray ionization preferences and the respective experimental work was limited by technical or methodological constraints, we systematically compared biochemical attributes of peptides identified with either instrument. This genome-wide, comprehensive study revealed biases toward certain peptide properties for both MALDI-TOF-TOF-and LTQ-Orbitrap-based approaches. These biases are based on almost 13,000 peptides and result in a general complementarity of the two approaches that should be exploited in future experiments. One-dimensional gel-based liquid chromatography mass spectrometry (GeLC-MS) 1 is a well-established technique in life science. In combination with in vivo labeling approaches such as stable isotope labeling by amino acids in cell culture (SILAC) (1) or 15 N labeling (2), it allows the relative quantification of large numbers of proteins in a complex sample. Mass spectrometry (MS) measurements in such workflows are predominantly performed with electrospray ionization (ESI)-based mass spectrometers. Online coupling of a liquid chromatography (LC) system with fast MS spectra acquisition and high-mass-accuracy ESI instruments in conjunction with fractionation on both protein and peptide levels allows the analysis of very complex samples in a relatively short period of time (3). The identification and quantification of data can be done in an automatic or semi-automatic manner with a variety of well-established software packages (4).In proteomic research, matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-TOF) is mainly used for the analysis of non-complex protein samples that do not require fractionation on the peptide level by means of liquid chromatography. In particular, the analysis of single protein spots resulting from the twodimensional PAGE separation of complex samples is primarily carried out with this MS tec...

MALDI‐TOF and nESI Orbitrap MS/MS identify orthogonal parts of the phosphoproteome

“…In contrast to observation made in earlier studies, which mainly analyzed samples of low molecular complexity, our LC‐MALDI data did neither indicate an increase in basic amino acid frequency in general , nor a preference towards the occurrence of C‐terminal arginine in particular . We did also not observe a preferred detection of multiply phosphorylated or large phosphopeptides which has previously been reported .…”

“…D). Consistent with previous work , we did not detect significant differences in phosphopeptide length and hydrophobicity (Supporting Information Fig. 1D) indicating that neither chromatographic factors nor the efficiency of peptide fragmentation/detection by MS can explain the apparent orthogonality of LC‐MALDI and LC‐nESI.…”

“…Recent work from Hessling et al. , extended the comparison of the two ionization methods to a more global proteomic scale and the data generally confirmed the previously observed complementarity of nESI and MALDI. Parker et al.…”

“…As complementarity of MALDI and nESI for peptide identification in bottom up proteomics has been demonstrated for unmodified peptides already , we sought to investigate to which extent this phenomenon extends to phosphopeptides. To address this, we first treated human A431 cells with sodium pervanadate in order to boost tyrosine phosphorylation, then digested the proteome and performed phosphopeptide enrichment using IMAC column chromatography and subjected identical samples to LC‐MS/MS measurements on a SCIEX 5800 MALDI‐TOF/TOF and a nESI Orbitrap Velos (Fig.…”

“…Proteases with alternative and orthogonal cleavage specificities such as Asp‐N, Arg‐C, chymotrypsin, Glu‐C and Lys‐C are efficient means to increase coverage of the proteome and phosphoproteome . Similarly, it has been shown that proteomic sequence coverage can be extended when using an alternative ionization technique because nanoelectrospray ionization (nESI) and MALDI, favor different physicochemical properties of peptides for ionization . Much of the early work comparing the merits of the two ionization methods used samples of low complexity (i. e. numbers of peptides analyzed), thus limiting the insight that could be obtained to a few dominating effects.…”