Nano LC-MALDI

Hofmann, S., Gluckmaim, M., Kausche, S., Schmidt, A., Corvey, C., Lichtenfels, R., Huber, C., Albrecht, C., Karas, M., and Herr, W., Rapid and sensitive identification of major histocompatibility complex class 1-associated tumor peptides by nano-LC MALDI MS/MS, Molecular and Cellular Proteomics 4(12), 1888-1897, 2005. 

G. Lochnit and R. Geyer, An optimized protocol for nano-LC-MALDI-TOF-MS coupling for the analysis of proteolytic digests of glycoproteins, Biomed. Chromatogr., 18 (2004) 841-848. 

Personalized medicine Reverse-phase peptide separation (nano-LC-MALDI-TOF/TOF) Fibronectin, kininogen-1, cartilage acidic protein 1, cartilage oligomeric matriz protein (61)... 

Benkali K, Marquet P, Rerolle JP, Meur YL, Gastinel LN. A new strategy for faster urinary biomarkers identification by Nano-LC-MALDI-TOF/TOF mass spectrometry. BMC Genomics. 2008 9(1) 541. 

Table 2. Comparison of the identified a-crystallin A peptides using nano-LC-ESI-MS/MS or nano-LC-MALDI-MS/MS, respectively.

The peptides exclusively identified by nano-LC-ESI-MS/MS are marked red, those detected by nano-LC-MALDI-MS/MS are labeled blue. 

Fig. 17 A new analytical strategy for comparing protein mixtures. Three protein samples, for example, obtained from one affinity pull-down and two control experiments, are compared with the objective of identifying the proteins that are only present in one of them, e.g., the affinity pull-down isolate. For this purpose, the latter is incubated with trypsin in H2 0. The two controls are pooled, then split into two parts, which are incubated separately with trypsin, one part in H2 0 and the other in H2 0. The resulting peptide mixtures are mixed in the ratio 3 1 2 and analyzed by nano-LC-MALDI MS and MS/MS. Tryptic peptides of proteins present only in the control samples or in the control samples and the affinity pull-down isolate are detected as paired signals with a distance of 4 Da, while peptides detected only in the affinity pull-down isolate are detected as unpaired signals. Reproduced from [316] with permission from American Chemical Society, 2006...

The complementary nature of MALDI-MS and ESI-MS-MS can also be exploited to increase the proteome coverage via PSA [36]. A fraction (20%) of the column effluent of a nano-LC column was sent to ESI-MS-MS, while the rest was fractionated onto MALDI spots for automated off-line LC-MALDI-MS and MS-MS analysis. About 63% overlap in protein identified from a digest of mammalian mitochondrial ribosomes was observed, but unique proteins were also identified by either technique. 

Based on a comprehensive shategy for the LC-MS characterizahon of membrane proteins [60], the analysis of in-vivo phosphorylated plasma membrane proteins from Arabidopsis thaliana was reported [40]. Phosphopephde isolahon was performed using SCX-IMAC, characterizahon with MALDI-MS and nano-LC-MS on a Q-TOF instrament. Six of the idenhfied sequences originated from different isoforms of plasma membrane H -ATPase, among which two new sites at the regulatory C-terminus. 

Ktister, S.K., Pabst, M., Jefimovs, K., Zenobi, R., Dittrich, P.S. (2014) High-resolution Droplet-based Fractionation of Nano-LC Separations onto Microarrays for MALDI-MS Analysis. Anal. Chem. 86 4848-4855. 

In conclusion, nano-LC combined with further improvements in MS sensitivity and speed will continue to reduce whole proteome analysis time for microbial strains by producing tens of thousands of peptide sequence-to-spectrum matches (PSMs) in less than 1 h (Hebert et al. 2014). However, LC-MALDI-MS/MS analyses may be better suited for specific applications requiring sample archiving. 

This results in co-crystaUization of matrix and sample and therefore higher sample concentration focused at the distinct hydrophilic anchor points [16]. One of the most commonly used matrices is a saturated solution of a-cyano-4-hydroxy-cinnamic acid in 97% acetone/3% water containing a small amoimt of TFA to support the ionization of the sample molecules in the MALDI source. The matrix is directed to the AnchorChip target and this is then dried. The sample fractionation after HPLC can be performed automatically using special sample fractionation systems (e.g., Probot, LC Packings Dionex). The target is introduced to a special desk and can be adjusted in the three XYZ planes so that the small nano-LC volume is placed directly onto the matrix. Using this set-up, fractions of volume less than 40 nL can be collected automatically. After collection, the individual fractions are recrystallized from 0.01% TFA, 30% acetone, and 60% ethanol in water and analyzed. 

Fig. 7. Application in practice. The spots of both gels are cut out after 2D-PAGE and Coomassie G-250 staining. The protein spots of one gel are analyzed by on-line nano-LC-ESI-MS/MS after tryptic digestion (a), whereas protein spots of the other gel are analyzed using off-line nano-LC and MALDI-TOF-MS/MS analysis (b).

With respect to resolving power (theoretical number of plates) and separation time, no doubt, capillary electrophoresis (CE) is the ultimate separation technique for complex peptide samples, and its combination with ESI MS online as well as MALDI MS off-line has been demonstrated many times [222-229]. The main reason why CE-MS, in contrast to nano-LC-MS, has not become a widespread method for protein and peptide analysis is the maximum total sample volume that can be separated by CE. In contrast to nano-LC, where many himdred microhters of dilute sample can be loaded without compromising separation power, the performance of CE directly depends on the sample volume and works best if only 50 nL or less is loaded. Recently, however, it has been reahzed that this requirement of CE is perfectly matched by nano-LC, which provides efficient sample concentration, and that the two techniques can be combined online upfront ESI or MALDI MS. For this purpose, a microfluidic chip was developed that enables, on demand, on-hne transfer (loading) of nano-LC fractions to an orthogonal CE separation channel, the effluent of which is either analyzed online by ESI MS or off-line by MALDI MS [230-232]. 

With this setup, nano-LC provides a first dimension of separation dining which the sample molecules are concentrated from a large volume, e.g., a few hundred microliters, to only a few hundred nanoHters or less. Each LC peak or a fraction of it is then separated in a short time (a few seconds) by CE and afterward analyzed by MS and MS/MS. The above 2D combination of nano-LC and CE in a chip format ready to combine with ESI or MALDI MS is a new development that has raised considerable interest. For the time being. 

In contrast to MALDI, the coupling of ESI with LC is far less challenging. Nevertheless, there are some problems that have to be considered. As already mentioned, polymers have to be dissolved in solvents suitable for the ESI process. Often catio-nization has to be promoted by an addition of salts to the solvent. Furthermore, the quantity of solvent that could be vaporized is limited to only a few microliters per minute, meaning that split techniques have to be adapted for ordinary LC apparatus or miao (or even nano) LC instmments have to be used. To overcome this limitation, micro-fabricated silicon chip-based ESI sources were developed. These chips consist of set of parallel etched channels, each with a width of 60 pm and a depth of 25 pm. 

The online or offline LC-ESI analysis also generated a list of peptide masses which were usually complementary to the MALDI peaklist. The few microliters left from the collected fractions after MALDI-MS analysis can be used, when necessary, for complementary analysis (MALDI-MS-MS or nano-ESI-MSn). 

The study of protein structure, function, quantity, and interactions during maturation and progression of disease is referred to as proteomics. Analytical approaches that use a combination of two-dimensional (2-D) gel electrophoresis for protein separation and MS analysis for protein identification followed by database searches is a widely practiced proteomics strategy.The tryptic peptides extracted from gels are analyzed by MALDI-TOF MS and microcolunm or capillary LC tandem mass spectrometry (MS/MS) techniques. Typically, the MALDI-TOF MS techniques are used to quickly identify peptide fragments and confirm the presence of known proteins. Nano-scale capillary LC/MS/MS techniques (using 50-100 pm diameter columns, operating at flow rates of 20-500 nL/min) are... 

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